International Congress BIOLOGICAL PRODUCTS - Gruppo di ...

International Congress 

BIOLOGICAL PRODUCTS: WHICH GUARANTEES FOR 

THE CONSUMERS 

Main Sponsors 

SIPCAM SpA, Milano 

BioTecnologie B.T., Todi PG 

Fondazione CARIPLO, Milano 

Milan 

October 15-16th 2002 

Congress Centre CARIPLO 

Via Romagnosi 6 

Milan 

Acknowledgement 

We are grateful to the CARIPLO Foundation for 

providing their prestigious Congress Centre 

Co-sponsors 

Università di Milano 

Assometab, Cesena 

Shimadzu Italia, Milano 

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Tuesday October 15 th 

9:30- 9:45 Registration 

9:45-10:15 Opening: 

Programme 

M. Trevisan (GRIFA president, University of Piacenza), 

L. Villa (Chairman of the Faculty of Pharmacy, University of Milan), 

On. L. Bellotti (Responsible of Biological Committee, MiPAF), 

M. Cocucci (Chairman of the Faculty of Agriculture, University of Milan), 

P. Cabras (University of Cagliari) 

SESSION 1 – Chairmen: M. Trevisan, E. Conti 

10:15-10:55 V. Rotondo 

FIAO, Italy 

The situation of organic farming in Italy: legislation and need for research 

10:55-11:15 Coffee break 

11:15-12:00 J. Casida 

University of California, Berkeley, USA 

Botanical insecticides: reflections and perspectives 

12:00-12:20 Y. Siderer, E. Anklam 

European Commission JRC, Geel, Belgium 

Need for research and appropriate analytical tools for organic food control 

12:20-12:40 C. Tuberoso 

Università di Cagliari 

Multiresidual methods for the determination of vegetable extract residues in 

products from organic farming 

12:40 -13:00 R. Lo Curto, F. Vilasi, T. Pellicanò, P. Munafò, G. Dugo 

Università degli Studi di Messina 

Presence of ochratoxin in experimental wines related to pesticides treatments 

employed on grapes 

13:00-14:30 Lunch 

14:30-15:00 Poster session 

SESSION 2 – Chairmen: P. Cabras, M. Solfrizzo 

15:00-15:40 C. Regnault-Roger 

Université de Pau et des Pays de l'Adour, France 

Prospects for new botanical insecticides: the example of Mediterranean 

aromatic plants. 

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2

15:40-16:20 G. Lozzia 

Università degli Studi di Milano 

Reliability and perspectives for the biological control of phytophagous insects 

16:20-16:40 M. Kelderer, E. Elias 

Centro Sperimentale Laimburg, Bolzano 

Efficacy of Ranya speciosa as natural insecticide 


17:00-17:40 S.K. Reilly 

U.S. Environmental Protection Agency, USA 

Biopesticides and their registration in the United States 

17:40-18:00 M. Rubbiani 

Istituto Superiore di Sanità, Roma 

Biopesticides: evaluation process following the application of the directive 

91/414/CE 

18:00-18:20 A. Ragni 

BioTecnologie BT (Italy) 

Entomopathogenic nematodes in biological control: reality and prospectives 

20:00 Social dinner (SOCREA, via Cino del Duca 8) 

Wednesday October 16 th 

SESSION 3 – Chairmen: M. Taccheo Barbina, G. Lozzia 

9:00-9:40 L. Gullino, A. Garibaldi 

Università di Torino 

Crop defence from fungal pathogens in organic farming: problems, effective tools 

and perspectives 

9:40-10:20 M. Solfrizzo 

CNR, Bari 

Toxicity and analysis of micotoxins and their presence in some organic foodstuffs 

10:20-10:40 B. Beretta, C. Ballabio, F. Tacchini, A. Cattaneo, C.L. Galli, C. Gigliotti, P. Restani 

Università degli Studi di Milano 

Determination of the content of mycotoxins in commercial products from organic 

and traditional farming 



3

11:00-11:40 A. Evidente 

Università di Napoli Federico II, Portici 

The fungal phytotoxins and their potential role as herbicides in control methods 

against weeds 

11:40-12:00 A. Santomauro, G. Tauro, M. Sorrenti, F. Faretra 

Università di Bari 

Protection of grapevine from powdery mildew by using natural substances and the 

microbial antagonist Ampelomyces quisqualis 

12:00-12:20 A. Di Muccio 

Istituto Superiore di Sanità 

Experiences in the controls of pesticide residues in Italian products from organic 

farming 

12:20-12:30 M. Benuzzi 

ASSOMETAB 

BioControl agents for organic agriculture: a devoloping field without 

appriopriate rules 

12:30-13:00 G. Imbroglini 

MiPAF 

The commitment of MiPAF for organic farming 

13:00-13:10 Closing remarks: M. Trevisan, P. Cabras 


4

MAIN SPEAKERS 

• V. Rotondo FIAO, Italy 6 

• J. Casida University of California, Berkeley, USA 8 

• C. Regnault-Roger Université de Pau et des Pays de l'Adour, France 10 

• G. Lozzia Università degli Studi di Milano 11 

• S.K. Reilly U.S. Environmental Protection Agency, USA 18 

• L. Gullino Università di Torino 19 

• M. Solfrizzo CNR, Bari 20 

• A. Evidente Università di Napoli Federico II, Portici 21 


5

LA SITUAZIONE DEL BIOLOGICO IN ITALIA: ASPETTI NORMATIVI E FABBISOGNO DI RICERCA 

Vincenzo Rotondo 

In rappresentanza FIAO 

C/o Suolo e Salute S.r.l., P.zza Mazzini, 42 Nettuno di Roma 

Ad oggi il territorio italiano offre la maggiore produzione da agricoltura biologica dell’Unione Europea. Il trend di 

crescita è sempre in aumento sia per le richieste del consumo che in offerta da parte della produzione. Dall’elaborazione 

dei dati forniti dagli Organismi di controllo operanti in Italia risulta che gli operatori del settore sono passati dai 54.004 

del 2000 ai 60.509 del 2001, suddivisi in 56.440 produttori agricoli ( di cui 1.600 produttori/trasformatori), 3.947 

trasformatori e 122 importatori. 

La distribuzione degli operatori sul territorio nazionale vede il 65% nel sud del paese, il 23% nel centro, il 22 nel nord. 

Per quanto riguarda la loro presenza nelle diverse aree geografiche delle attività produttive, si rileva la prevalenza al sud 

dei produttori agricoli (68%, contro il 20% al nord e il 12% al centro), ed al nord dei trasfo rmatori(47% contro il 19% al 

centro ed il 34 al sud), e degli importatori (82% contro l’11% al centro ed il 7% al sud). 

La superficie interessata, in conversione o interamente convertita ad agricoltura biologica risulta pari a 1.237.640 ettari, 

pari all’8% circa della SAU. Le principali colture riguardano i foraggi ed i cereali (221.436 ettari), i prati e pascoli 

(241.157 ettari), che nel loro insieme rappresentano il 70% circa degli investimenti. Seguono in ordine di importanza le 

coltivazioni arboree (olivo, vite, agrumi e frutta) con il 20% e le colture orticole ed industriali (leguminose da granella, 

prodotti orticoli, colture industriali) 4%. 

Per le produzioni animali, distinte sulla base delle principali tipologie produttive, al 31.12.2001, si segnala la seguente 

situazione: bovini 330.701 (latte e carne), ovi-caprini 327.891, pollame 648.693, conigli 1.682, api, in arnie, 48.228. 

L’attività di controllo, esercitata dagli organismi autorizzati dal Ministero delle Politiche Forestali e dalla Provincia 

Autonoma di Bolzano, si è concretizzata in 72.896 visite ispettive, prelevamento ed analisi di 7.332 campioni. L’attività 

di controllo ha portato al rilevamento di 2.074 irregolarità ed all’applicazione, in via definitiva, di 1.367 sanzioni. 

Numero operatori suddivisi per regione ed attività (dati provvisori) 

REGIONE 

N. OPERATORI CONTROLLATI 

P T I 

6 

TOTALE GENERALE 

ABRUZZO 942 113 2 1.057 

BASILICATA 656 33 - 689 

CALABRIA 7.087 131 - 7.938 

CAMPANIA 1.782 174 4 1.960 

EMILIA ROMAGNA 4.535 531 39 5.105 

FRIULI VENEZIA 

243 58 1 302 

GIULIA 

LAZIO 2.415 225 - 2.640 

LIGURIA 314 65 4 383 

LOMBARDIA 1.023 379 23 1.425 

MARCHE 1.807 129 2 1.938 

MOLISE 476 34 - 510 

PIEMONTE 3.250 312 12 3.574 

PUGLIA 6.470 361 3 6.834 

SARDEGNA 7.798 88 - 7.886 

SICILIA 12.225 424 - 12.649 

TOSCANA 1.923 318 7 2.248 

TRENTINO ALTO 

551 97 2 650 

ADIGE 

UMBRIA 948 81 4 1.033 

VALLE D’AOSTA 18 2 - 20 

VENETO 1.257 392 19 1.668 

TOTALI 56.440 3.947 122 60.509 


Il settore oggi è regolamentato in ambito comunitario con i Regolamenti 2092/91 con tutte le integrazioni e 

modificazioni successive ed il Regolamento 1804/99. In ambito nazionale il D.L. 220/95 è il riferimento del 

recepimento nazionale alla normativa comunitaria. 

A causa dello sviluppo consistente e della complessità nell’offrire la più ampia garanzia al consumo risulta necessario 

revisionare la normativa al fine di modificare parte dei contenuti, sia a livello nazionale attraverso una legge delega che 

in considerazione della grande opportunità in prospettiva alla potenzialità del suo territorio, valorizzi ed incentivi le 

produzioni biologiche, sia a livello comunitario in modo tale da razionalizzare e contemplare in un’unica disciplina di 

settore la materia del biologico, determinando anche maggiore chiarezza, armonizzazione tra i vari comparti 

dell’agroalimentare , creare i giusti riferimenti per l’informazione, la promozione, la progettazione ed in modo 

particolare la ricerca. 

Grande assente ingiustificata è la ricerca nel settore dell’agricoltura biologica. 

Nel nostro paese la sperimentazione a supporto del metodo biologico è piuttosto limitata e trascurata dagli enti preposti, 

salvo rare eccezioni dovute a volontà individuali. Si registra altresì, uno scollegamento fra le realtà produttive e le unità 

di ricerca. Molti problemi di entità apparentemente complessa possono essere risolti attraverso lo sviluppo di attività 

sperimentali specifiche , da osservazioni e prove accurate che abbisognano di studio e tecnologie appropriate. 

Mancano fondamentalmente alternative valide per sostituire i prodotti rameici, conferme sull’utilizzazione di 

determinati principi attivi e corretto impiego, soluzioni ai problemi tecnici delle nostre aree come il risanamento di 

terreni inquinati dalle sostanze chimiche utilizzate decenni fa (clororganici), individuazione degli antagonisti dei 

parassiti e loro corretto impiego nella lotta biologica, incremento dello studio sulle foraggere proteiche sia per il 

fabbisogno zootecnico che come miglioratrici dei suoli, proteggere la biodiversità, aumentare l’attenzione sull’uso 

indiscriminato di sementi OGM, ecc. Il settore resta in attesa di interventi a favore di queste attività che porterebbero 

indiscutibilmente giovamento a chi produce e stimolo per chi vuole iniziare. 


7

BOTANICAL INSECTICIDES: 

REFLECTIONS AND PERSPECTIVES 

John E. Casida 

Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, 

University of California, Berkeley, California 94720-3112, USA 

Importance and History. Insects are our main competitors for a limited supply of food and fiber. Pests destroy 

about one-third of the world food supply during growth, harvesting and storage, even with the use of pesticides and 

other control measures. The insecticides have evolved from inorganics to botanicals to synthetic organics. The inorganic 

insecticides have almost disappeared from use on crops for reasons of inadequate effectiveness and safety. The 

botanicals and synthetic organics have many similarities yet important differences and limitations. The author presents 

some reflections and perspectives on these relationships from a half century of personal experience in insecticide 

research. 

Five Major Botanicals. There are five major botanical insecticides, i.e. pyrethrum, rotenone, nicotine, neem 

and ryanodine. Pyrethrum extract from the flowers of the white daisy Chrysanthemum cinerariaefolium is the most 

important. The countries of production include Kenya, Tanzania and Australia. The effectiveness is enhanced and the 

economics improved by adding piperonyl butoxide for stabilization from metabolic detoxification in insects. Pyrethrum 

is also important because its active ingredients, the pyrethrins, provided the model for the synthetic pyrethroids which 

now constitute 20% of the market value of all insecticides. Rotenone from the roots of Derris elliptica and 

Lonchocarpus utilis has for 150 years been used to control chewing insects and as a piscicide to kill unwanted fish. 

Rotenone has been the prototype for Complex I respiratory inhibitors but not for economically successful structural 

modifications. Home gardeners recommend rotenone use. Nicotine, the alkaloid of tobacco plants (and cigarettes), has 

declined in use for decades but serendipitously served as the mode of action but not structural prototype of the new 

synthetic neonicotinoids of high effectiveness and apparent safety. Neem has important local uses in India and China 

and the extract with azadirachtin active ingredient is an effective crop protectant acting as an antifeedant. Ryania is the 

ground stemwood of Ryania speciosa. The supply is limited but the effectiveness is high for lepidopterous larvae. 

Supply and Composition. One of the limitations of botanical insecticides is consistent supply with adequate 

standards of effectiveness and safety. This is a constant challenge even with the best producer organizations. Pyrethrum 

once had an allergen now removed by a defined cleanup procedure. We analyzed cubé resin (the commercial rotenone 

supply) and found 11 stilbenes and flavonoids and 29 rotenoids (including one that incorporated chlorine from the 

solvent extraction conditions). Nicotine has been extensively studied except perhaps its environmental degradation 

products. Neem contains many active ingredients; we described nimbolide and epoxyazadiradione as cytotoxic agents. 

The principal active ingredient of Ryania was long described as ryanodine until we found that 9,21-dehydroryanodine 

was of even greater importance. 

Toxicology. Botanicals are more difficult to evaluate toxicologically than synthetic insecticides. They usually 

have one or two major active ingredients (e.g. pyrethrins I and II in pyrethrum extract) but there may also be many 

related bioactive compounds (e.g. in neem). Unless highly purified they also have hundreds to thousands of other 

extractives or natural products which can vary with the plant source and processing. Critical evaluation of toxicology 

requires production and use samples of consistent composition. Toxicology studies on pyrethrum extract meet current 

standards but those for other botanicals are lacking in scope, although this does not necessarily mean hazards are 

involved. 

Residues of botanical insecticides are rarely a known problem. The pyrethrins photodecompose rapidly to 

compounds of greatly reduced neurotoxicity. The same applies to the rotenoids and ryania components. Natural 

products are generally considered to be easily metabolized. They have few or no halogens or refractory substituents. 

Quite the contrary they have phenol, alcohol and ketone substituents, and alkene moieties that are amenable to ready 

degradation. The analyses are more difficult than with most synthetics: the lack of halogens limits the analytical 

detector sensitivity; multiple components and related but inactive analogs complicate the process. 

Mode of Action and Resistance. Mode of action is an important aspect of insecticide use, safety and 

toxicology. The same targets are involved in the most part with the botanicals and synthetics. Major insecticides act on 

four principal targets in the nervous system. 


8

target botanical synthetic 

sodium channel – pyrethrins DDT, pyrethroids 

voltage dependent 

chloride channel - picrotoxinin* cyclodienes, lindane, 

GABA-gated fiproles 

acetylcholinesterase physostigmine* organophosphates 

methylcarbamates 

nicotinic receptor nicotine neonicotinoids 

*never a major commercial insecticide 

The synthetics indicated above constitute about 90% of the insecticide market. This has major implications in the 

selection for resistance leading to higher required doses and less effective control. Selection with the synthetics can lead 

to target site resistance to the botanicals, e.g. the use of synthetic pyrethroids and even DDT results in selection of 

strains resistant to pyrethrins. 

Prototypes. Botanicals and other natural products are primary sources and prototypes in the search for new 

insecticides. Screening of plant extracts enables the search of thousands of compounds as mixtures. This can be done at 

random or with knowledge of potential usefulness from practice or folklore. Bioassay-directed isolation and modern 

characterization techniques can often lead to a defined structure in weeks. This practice for a half century has turned up 

potent compounds usually with problems of inadequate selectivity for insects versus mammals. The variety and source 

of organisms are not infinite and limitations in this approach are already evident in the number of new isolates that 

turned out to be previously known components with insecticidal activity. Medicinal plants and herbs are frequently used 

or proposed to control insect pests. A literature search will usually reveal the active ingredient and often the mode of 

action. Isolation of the effective component will usually verify that one is using a new source of an old control 

chemical. The approach is still good but with increasing limitations. 

Botanicals are more likely than synthetics to give leads for completely new types of insecticides. With 

synthetics, the problem is to generate a new lead compound and then prepare massive numbers of analogs and rapidly 

optimize their structure. Combinatorial procedures help here. Fast throughput screening allows rapid sifting for 

effectiveness and mode of action. 

Organic Agriculture. Botanical insecticides and natural foods fit well together. Rapidly increasing knowledge 

helps choose the best materials and insures their effectiveness and safety. The natural chemical defenses of plants used 

as botanical insecticides offer continued benefits in organic agriculture with confidence that the consumer and 

environment will be adequately protected. 


9

PROSPECT FOR NEW BOTANICAL INSECTICIDES : 

THE EXAMPLE OF MEDITERRANEAN AROMATIC PLANTS 

Catherine Regnault-Roger 

Laboratoire d’Ecologie Moléculaire 

Université de Pau et des Pays de l’Adour-France 

BP 11 55 – F-64013 PAU Cedex 

catherine.regnault-roger@univ-pau.fr 

Fifty years of sustained struggles against harmful insect using chemical synthetic molecules has produced perverse 

secondary effects like mammalian toxicity, insect resistance ans ecological hazards. Now, IPM has to face up to the 

economic and ecological consequences of the use of pest control measures. The diversification of the approaches 

inherent to IPM is necessary for friendly environnemental management. Among the alternative strategies, the use of 

plants with insecticidal allelochemicals appaer to be promissing. The developpement of this ecochemical approache 

requires the identification of efficient extracts or compounds from new sources of tropical but also temperate plants. 

Aromatic plants are abundant on the Mediterranean periphery and they have been used as spices, pot-herb or medicinal 

plants since Antiquity. Numerous industrial applications have then been implemented in perfumery, cosmetics and 

detergents, pharmacology and fine chemistry as well as aromatics for the food industry. A recently-appeared field, IPM, 

may also be prospected as an application field for aromatic Mediterranean plants: they contain a large range of 

allelochemicals, which develop insecticidal properties. These insecticidal activities could be considered as 

complementary or renewal strategies in IPM. 

In Southwestern of France, the use of aromatic plants as traditional protectors of stored products is an old custom. We 

evaluated in our laboratory the efficiency of these traditional customs: we observed that some plants were insecticidal, 

especially those belonging to Lamiaceae. Thymus spp, Origanum spp, Rosmarinus sp were among the most active. 

Our first insect model was Acanthoscelides obtectus Say (Bruchidae, Coleoptera) and its host plant Phaseolus vulgaris, 

Leguminosae. HPLC analyses were conducted to identify main components and bioassays to determine their biological 

activities on the beetle. The toxicity on adults and the inhibition of insect reproduction were evaluated. Essential oils 

were active, however not exclusively as some hydrodistillated residues also exerted a toxic activity. The main active 

compounds were identified as monoterpens and polyphenols. 

Then, their effects on insects of different kinds like Ceratitis capitata (Diptera), Rhopalosiphum padi and 

Metopolophium dirrhodum (Aphids) or Lepidoptera were tested. These evaluations showed that a large variability could 

be observed in the response of different insect species to a same compound. Variability of plant chemical patterns 

induced by climatic, pedological and genetic variations was also noticed. 

According to these results, structure-activity relationships are discussed here in regard to their efficiency to control pest 

insects and to their suitability to be considered as potential biopesticides. 

References : 

C.REGNAULT-ROGER, B.J.R. PHILOGENE, C.VINCENT, 2002 : Biopesticides d’origine 

végétale, Lavoisier (Paris), pp 337. 

C.REGNAULT-ROGER : The potential of botanical essential oils for insect pest control, Integrated Pest Managements 

Reviews, 2, 25-34 (1997). 


10

RELIABILITY AND PROSPECTIVES FOR THE BIOLOGICAL CONTROL OF PHYTOPHAGOUS 

INSECTS 

Giuseppe Carlo Lozzia, Ivo E. Rigamonti 

Istituto di Entomologia agraria, Università degli Studi di Milano 

In the last years there has been an explosion in the widespread of foodstuffs and other “biological” products that are 

associated with an image of naturalness and hygienic safety by the public, even if these concepts are wrongly thought to 

be closely linked. In reality, notwithstanding the great emphasis on the use of “natural” techniques and of the exclusion 

of artificial products, biological agriculture is not so different from the most modern “conventional” strategies, and is 

largely based on the use of active ingredients, which are natural but still toxic, that have all the problems of “pesticides” 

(residues on foodstuffs, side effects on non-target organisms, etcetera) but on a different scale. This situation, both for 

biological and traditional agriculture, is not due to a “strategic” trust in pesticides but to a lack of total efficacy in low 

impact management techniques, and in particular to biological control. 

Up to now, as a matter of fact, there is no culture of a certain importance for which the pest management can be given 

solely to, or even mainly to, biological control. The situation is decidedly better if we only refer to animal organisms. In 

this case there are consolidated strategies, at least theoretically; which refer to the reconstitution of balances and to the 

improvement of the action against natural enemies, which are carried out in practice through the use of four 

“techniques”: the diffusion of antagonists, the elimination of worrying sources and the adoption of environmental and 

cultural management measures. Furthermore, the use of pheromones and other “modern” methods gives the possibility 

to enlarge the possibilities of success. Modern strategies are based on this framework especially with the understanding 

that a culture is a real ecosystem where limiting natural factors have a considerably important role. The most recent 

research is analysing the influence of different cultural practices on the coenosys settled in the agro-ecosystem. The aim 

of all this is, on the one hand to favour the diffusion of techniques with beneficial effects, and on the other to limit the 

repercussions of harmful interventions which cannot be eliminated, as some phytosanitary treatments (Bassino, 1987). 

One of the areas where the use of biological control practices is widely used, is in glasshouses cultures. The quick 

succession of cultures one different from the other, with brief periods when there are no cultures, create a very 

particular environment where techniques of permanent biological control of phytophagous insects have no possibility of 

success. For this reason, there are usually routine “inundative” releases of antagonists in glasshouses and in tunnels. 

Using this strategy the biological tool is almost like a chemical treatment. Large quantities of predators or parasitoids 

are introduced into the structure in order to quickly bring down the population of the target pest and to keep it thereafter 

under the damage level until the end of the cultivation cycle. Often the release has to be repeated over and over again 

and more than one species of antagonists are introduced. In these environments phytophagous insects with determinate 

characteristics tend to become affirmed (polyphagous, high number of generations, very prolific) which are able to 

damage the cultures that follow, that, as already mentioned, can be very different one from another, and in any case the 

species always tend to be the same ones. Numerous Rhynchota are particularly abundant (whiteflies, aphids, scale 

insects), but Agromyzidae Diptera can also be easily found, as well as some Thysanoptera (Frankliniella occidentalis 

(Pergande), Heliothrips haemorrhoidalis (Bouché), Thrips tabaci Lindemann) and spider mites. The low number of pest 

species makes it possible for key antagonists to be restricted to a few units. All this has led to the birth and the diffusion 

of structures, the commercial insectary, that have become similar in organisation to an industry, whereby the most 

important natural enemies of every phytophagous insect is bred in large quantities and after put on sale on a large scale. 

The breeding of auxiliaries is very complex (figure 1) requiring the development of refined and very reliable 

techniques. 


11

Preparation of the medium 

(plant or diet) 

Figure 1 - Breeding process in a commercial insectary. 

Multiplication of the victim 

Multiplication of the auxiliary 

Collection 

Packaging 

Product quality control 

Delivery 

The high cost involved, together with the need to have a sure market has until now limited considerably the field of use 

to protected environments which, on the one hand, usually host highly precious and expensive cultures and, on the 

other, do not allow the dispersion of the antagonists, optimising their efficacy. Among the species to be found in 

breeding there are numerous Hymenoptera parasitoids as the Aphelinidae Encarsia formosa Gahan, active against 

whiteflies, or the Eulophidae Diglyphus isaea (Walker) which attacks the Agromyzidae Lyriomiza trifolii (Burgess), 

and among the predators the Neuroptera Crysopidae Chrysoperla carnea (Stephens) an enemy of many aphids and the 

Phytoseiid mite Phytoseiulus persimilis Athias-Henriot an antagonist of spider mites. To be fair, it has to be said that 

many micro-organisms, in a broad sense, are produced commercially for the same purpose as Bacteria (Bacillus 

thuringiensis Berliner), fungi (Beauveria bassiana (Bals.)) or Nematodes (Steinernema feltiae (Filipjev) and S. 

carpocapsae (Weiser)). 

For cultures to be found exclusively in the open field, an example of a nearly optimal situation can be supplied by 

viticulture, which in the last decades has led to the affirmation of different practices of biological control which cover 

all the kinds of strategies that have been mentioned above. This evolution has been made easier by the pre-existence of 

a remarkably complex arthropod coenosys and therefore highly stable (Boller and Remund, 1986; Boller and Basler, 

1987). The brief description of the different measures in the following paragraphs allows us to understand what are the 

lines of research and what is the path that makes the practical enactment possible. 

The first measure, the elimination of a worrying source, has been the rationalisation of the use of pesticides which, since 

the 80’s has helped to rebuild the populations of Phytoseiids, predator mites responsible for the biological control of 

Tetranychidae and Eriophyidae. It has been proved that the choice of products, in particular of fungicides are 

fundamental for the maintenance of Phytoseiids and since the effects tend to accumulate, even substances which are 

relatively little toxic can be extremely harmful if they are used repeatedly or in particular moments of the season. 

However, in answer to specific conditions it may be possible to use active toxic substances as dithiocarbamates, dinocap 

or powdery sulphur. In this case it is necessary to know the characteristics and the critical moments for the dominating 

species in the concerned area, since they are different in the various Italian regions. For example, Kampimodromus 

aberrans (Oudemans) is really sensitive and requires the use of the least harmful active ingredient possible from those 

on offer. Typhlodromus pyri Scheuten is more tolerant instead but has moments of high sensitivity when it has to be 

protected fully. In the more northern areas this period coincides with the regrowth of the vegetation, when there are a 

few females and in addition they are very “stressed” due to the adverse winter conditions. In the more southern areas 

this normally takes place in the summertime, as T. pyri does not like high temperatures very much. If done correctly, the 

effects can be limited to a selection pressure which favours the affirmation of the more resistant Phytoseiids without 

compromising their efficacy. The protection of these predators is furthermore enhanced by the support offered by the 

practices of habitat management aimed at the diffusion of tree areas. Here, populations of Phytoseiids settle undisturbed 

which interact with the culture thanks to the passive diffusion of individuals carried over by the air currents and which 

give the possibility to overcome critical moments through the repopulating of populations decimated by erroneous 

cultural practices or even by the recolonisation of the culture (figure 2). 

12 

breeding of the victim 


Stock

Vineyard 

Passive diffusion 

Spontaneous flora 

Figure 2 - Phenology of the movements of Phytoseiid populations in the grapevine agroecosystem in Northern Italy. 

A general presence of trees and shrubs is not the only requirement but well defined elements able to host the interesting 

species for the culture is. Therefore K. aberrans is abundant in our regions on many hosts (Celtis australis, Corylus 

avellana, Ficus carica, Fraxinus excelsior) (Duso et al., 1993; Coiutti, 1993) while T. pyri is important only on Rubus 

fruticosus and Sambucus nigra (Lozzia and Rigamonti, 1990) to be added to Acer spp., Cornus sanguinea, Corylus 

avellana, Lonicera xylosteum and Ulmus spp. in Switzerland (Boller et al., 1988). 

At a later stage, measures of environmental management have been proposed, that involve, in different periods of the 

season, both the natural vegetation and cultivation, able to obtain that Empoasca vitis Goethe be under the danger 

threshold. The evolution of the process has been long and hard. It has been known for a long time that E. vitis carries 

out its cycle on different hosts and hibernates on conifers and other evergreen plants different from those that it attacks 

(Vidano, 1958) and that, starting from the beginning of May, there is a mass migration from the plants where it 

overwinters towards the cultures as soon as they blossom. The plants that are involved in rapid succession are the 

following: firstly the apple tree, roses and some stone fruits then the grapevine, which seems to be the favourite plant on 

which most of the population concentrates (figure 3). 

13 

Trees 

Weeds 


ose, bramble, dogrose 

birch 

hazelnut 

apple tree 

vine 

Picea sp. 

March April May June 

Figure 3 – Phenology of Anagrus atomus, adults and eggs , and of Empoasca vitis, adults and 

eggs , on different host plants (from Cerutti et al., 1989, modified). 

The species stays here until summer when, from the beginning of August, the hibernating females which leave the 

vineyards start to appear. The first step has been to identify the key antagonist, identified as Anagrus atomus Haliday, 

an Hymenoptera Mymaridae, oophagous parasitoid that controlled “naturally” in some vineyards this leafhopper, being 

able to hit a part between 20 and 82% of the germs (Arzone et al., 1988; Vidano et al., 1988; Cerutti et al., 1990). Later 

on Cerutti et al. (1989) studied the cycles of E. vitis and of A. atomus highlighting the fundamental importance of the 

maintenance of plants on which the parasitoid spends the last stages of the season and passes the winter and above all, 

on which it starts again its activity in April just before the sprouting of the grapevine (figure 3). Therefore it is the adults 

of the first generation of the year that migrate on the culture starting from the end of May. The success of the 

colonisation of the vineyard by the parasite therefore depends on the presence of suitable host plants. They include 

different Rosaceae (Rubus spp., Rosa spp., the apple tree), Lonicera sp., Corylus avellana and also Betula pendula. In 

such structured areas the action of A. atomus on the leafhopper is decisive. Very similar results had been obtained in 

North America too, where the species involved were Anagrus epos Gir. and Erythroneura elegantula Osb.. In this 

way in numerous cases the permanent control of these harmful leafhopper in different European countries and in North 

America has been obtained. At present, though, the diffusion of these strategies is obstructed by too low threshold 

levels which over-estimate the symptoms, such as the leaf reddening, which probably are of little importance. As a 

matter of fact the threshold level is 2 or even 1 specimen per leaf while it has been noted that there are no effective 

damages even if there are more than 3 specimens per leaf (Lozzia and Rigamonti, 1994). Also considering the different 

sensitivity of the cultivars it would be a good idea to increase these threshold levels in order to avoid that useless 

treatments reduce parasitoid populations, obviously where the environmental conditions described above exist or where 

it is thought they will start. In this general framework Jacobiasca lybica (Bergevin & Zanon) is not included. In Italy it 

is mainly to be found in Sardinia, it is harmful at lower densities than with respect to the preceeding species and does 

not seem, at present as far as is known, to be sufficiently controlled by natural antagonists. 

In the last years a programme of inoculative releases has been started in many regions, whose aim is to rebuild the 

balance between an exotic phytophagous insect, the Rhynchota Flatidae Metcalfa pruinosa (Say) and one of its 

parasitoids, the Hymenoptera Dryinidae Neodryinus typhlocybae (Ashmead). In this case, too, the introduction of the 

auxiliary in the environment is the final result of a long research process. The leafhopper arrived in Italy from Northern 

America at the end of the 70’s (Zangheri and Donadini, 1980) and since then it has spread in different European 

countries. Thanks to the fact that it is polyphagous it has colonised the most diverse environments, including many 

cultures to which it is harmful, especially due to its abundant production of honeydew and to the problems linked to 

this. In the first years after its arrival, the research for its natural enemies in the area of origin was carried out and the 

key antagonist N. typhlocybae was identified. After this a breeding on a small scale of this Dryinidae was started in 

order to evaluate its capabilities in the laboratory and with the specimens obtained the first experimental tests were 

carried out in the field. After having obtained a mass production, the launch on a large scale was started at the end of the 

90’s (Girolami et al., 1996). The technique is the classic one adopted in these cases. Taking advantage of the polyphagy 


14

of the host, a relatively limited number of parasitoids is introduced on natural plants surrounding a plot. In this way the 

auxiliaries can multiply without the risk of being killed by phytosanitary treatments normally carried out on the 

grapevines. Once the population has reached a sufficient density level a flow of individuals begins and reaches the 

culture and settles there; if there are no harmful interferences the colonisation is permanent but even if this is not so the 

“reservoir” of the population on natural plants can guarantee a remarkable level of protection. The critical moments in 

these cases are usually the capability of the antagonist to adapt to the new environment, in particular the overcoming of 

the bad season, and its diffusion potential on a large radius starting from the introduction points. At the present moment 

there are no definitive results as yet as to the success of this project. 

A last example, which includes the combination of more measures, is the control of the grape berry moths Lobesia 

botrana (Den & Schiff.) and Eupoecilia ambiguella (Hb.). The first solution is the progressive substitution of normal 

treatments carried out with pesticides with techniques with a progressively lower impact on the environment, as the use 

of growth regulators, of microbiological insecticides and lately with mating disruption. This last strategy uses, as is 

known, large quantities of sexual pheromones whose aim is to “confuse” the males making them incapable of 

distinguishing the traces secreted by the females, thus not allowing the coupling and as a consequence the reproduction. 

This solution, optimal from the environmental point of view, has different “technical” limitations though, thus obtaining 

the best results only if it is used on large and homogenous areas and in the presence of low populations. This last 

constraint has led in the past different Authors to suggest to bring down the moths populations with ad hoc treatments 

and this has not allowed the use of mating disruption in many areas in Italy. Together with this, and to eliminate these 

limitations in part, habitat management measures are being introduced, whose aim is to improve the action of the 

natural antagonists of the moths, in particular of the parasitoids. Since the majority of these species in the adult stage is 

glyciphagous, it has been very important to guarantee them the presence of a suitable source of food, which is made up 

of nectar plants in flower within or near the vineyard. The critical points are therefore two: on the one hand the presence 

of species that produce nectar and are attractive for the parasitoids and, on the other hand, the continuous presence of 

plants in flower. This can be obtained by sowing suitable species and with the alternate mowing of the rows. 

These are clear examples of the holistic approach, which is typical of the most recent evolutions in cultural management 

strategies. The control of a phytophagous insect is obtained by putting together different techniques both preventive and 

curative, which involve different aspects of its biology and of its relations with the rest of the agroecosystem. 

Furthermore, practices such as weeding and alternate mowing have a more complex meaning. They allow to maintain a 

continuous presence of flowers, which are a fundamental source of food for many useful or indifferent arthropods 

(figure 4), and they allow not only the increase of the number of their species but also the presence of denser and more 

stable populations (Remund et al., 1989; Remund et al., 1992). 

Nectar for Lepidoptera 

Nectar for parasitoids 

Urtica dioica X X X X 

Lamium maculatum X X X X 

Daucus carota X X X 

Aegopodium podagraria X X X 

Galium mollugo X X X X 

Origanum vulgare X X 

Rosa canina X X X X X 

Rubus fruticosus X X X X X 

Lonicera xylosteum X X 

Figure 4 - Ecological meaning of the flora between the lines and in the adjoining areas of the vineyard with respect to 

the antagonists of grape berry moths, their alternative hosts and other auxiliary species. 

This data makes us suppose that in these environments a regulation of the harmful insects and mites at low population 

levels and a stability of the whole agroecosystem is possible. Numerous species of parasitoids and predators which find 

a great number of alternative hosts or preys among the insects and mites to be found on flowers and grass are 

favourably hit by this presence. The pollen of anemophile species, instead, when it reaches the grapevine, is an 


15 

Oophagous parasitoids 

Larval parasitoids 

Pupal parasitoids 

Predatory mites 

Pollen for predatory mites 

Oophagous parasitoids of 

leafhoppers

important alternative foodstuff for the Phytoseiids (Wiedmer and Boller, 1990; Remund and Boller, 1992; Engel and 

Ohnesorge,1994) that are able to colonise even the grass flora, in particular many dicotyledons that then become 

another reservoir of useful species available for the grapevine (Lozzia and Rigamonti, 1998). 

Biological control, as can be seen from the above examples and from numerous other cases, has often been an 

efficacious and reliable instrument for a permanent solution linked to infestations of single phytophagous insects. 

However, it still cannot be considered suitable for the management of pests on the whole, not even limiting itself to 

animal organisms. Also in the case of the wine grapevine, which is perhaps the culture that today has the best situation, 

there have been problems following the expansion of flavescence dorée, a phytoplasma disease carried by Scaphoideus 

titanus Ball, whose control requires the carrying out of treatments against the vector, interventions that risk to frustrate 

also the results obtained with biological control against other phytophagous insects of the grapevine. This situation can 

be generalised and applied to all viral, bacterial or phytoplasma diseases transmitted by vector organisms, whether they 

be insects, mites or nematodes. In these cases, in fact, it is necessary to maintain the populations of the parasite at 

extremely low density levels that cannot be obtained through the action of natural antagonists which for their own 

existence, need the presence of a suitable quantity of victims. Only the availability of resistant plants will make 

treatments against vectors superfluous in the future. 

The management of vector species, the one of key phytophagous insects of the various cultures for which usually 

“natural” control is not sufficient, the side effects of fungicide treatments are at present and in the near future the main 

obstacles to the creation of a phytosanitary defence against phytophagous insects only using biological control 

strategies. Another factor that must be considered and that today limits the reliability of biological control is the 

dynamic nature of agroecosystems. The appearance of new phytophagous insects, the pullulations of secondary or 

occasional species, the expansion of the area of numerous pests and so on, are highly probable events but they cannot be 

forecasted, and this makes it necessary to have continuous adjustments to the methods adopted even if they are widely 

experimented and to study new strategies. The same variability that exists between different areas whereby a culture is 

diffused implies the need not to generalise solutions but to find those more suitable to the single realities through a 

continuous process of experimentation. To sum up, we can say that biological control is without a doubt the main 

weapon on hand in modern agriculture for the management of phytophagous insects but it still requires many studies 

before all its potential can be used to the full and for a certain period of time it will be necessary to use more invasive 

techniques. 

BIBLIOGRAPHY 

ARZONE A., VIDANO C., ARNÒ C., 1988 - Predators and parasitoids of Empoasca vitis and Zygina rhamni 

(Rhynchota Auchenorrhyncha). In Vidano C. & Arzone A. (ed.), Proc. 6th Auchenorrhyncha Meeting. CNR- 

IPRA, Torino: 623-629. 

BASSINO J.P., 1987 - Principles of integrated protection in vine growing. A.A. Balkema Ed., Rotterdam, Proc. 

Meeting E.C. Experts’ Group “integrated Pest Control in Viticulture”, Portoferraio, Italy, 26-28/9/1 985: 329- 

336. 

BOLLER E., REMUND U., 1986 - Der Rebberg als vielfältiges Agro-Oekosystem. Schweiz. Z. Obst-Weinbau, 122 (2): 

45-50 

BOLLER E., BASLER P., 1987 - Pflanzenschutzmassnahmen in Weinbau im Rahmen der integrierte Produktion. 

Schweiz. Z. Obst-Weinbau, 123 (2): 61-63. 

BOLLER E.F, REMUND U., CANDOLFI M.P., 1988 - Hedges as potential sources of Typhlodromus pyri, the most 

important predatory mite in vineyards of northern Switzerland. Entomophaga, 33: 249-255. 

CERUTTI F., DELUCCHI V., BAUMGÄRTNER J., RUBLI D., 1989 - Ricerche sull’ecosistema “vigneto” nel Ticino: 

II. La colonizzazione dei vigneti da parte della cicalina Empoasca vitis Goethe (Hom., Cicadellidae, 

Typhlocybinae) e del suo parassitoide Anagrus atomus Haliday (Hym., Mymaridae), e importanza della flora 

circostante. Mitt. Schweiz. Ent. Ges., 62: 253-267. 

CERUTTI F., BAUMGÄRTNER J., DELUCCHI V., 1990 - Ricerche sull’ecosistema “vigneto” nel Ticino: III. 

Biologia e fattori di mortalità di Empoasca vitis Goethe (Homoptera, Cicadellidae, Typhlocybinae). Mitt. 

Schweiz. Ent. Ges., 63: 43-54. 

COIUTTI C., 1993 - Acari Fitoseidi su piante arboree spontanee e coltivate in Friuli Venezia Giulia. Frustula entomol., 

n.s. 16: 65-77. 

DUSO C., TORRESAN L., VETTORAZZO E., 1993 - La vegetazione spontanea come riserva di ausiliari: 

considerazioni sulla diffusione degli Acari Fitoseidi (Acari Phytoseiidae) in un vigneto e sulle piante spontanee 

contigue. Boll. Zool. agr. Bachic., Ser. II, 25: 183-203. 

ENGEL R. VON, OHNESORGE B., 1994 – Die Rolle von Ersatznahrung und Mikroklima im System Typhlodromus 

pyri Scheuten (Acari, Phytoseiidae) – Panonychus ulmi Koch (Acari, Tetranychidae) auf Weinreben. 1. 

Untersuchungen im Labor. J. Appl. Ent., 118: 129-150. 

GIROLAMI V., CONTE L., CAMPORESE P., BENUZZI M., MARTIR G. R., DRADI D., 1996 - Possibilità di 

controllo biologico della Metcalfa pruinosa. Inf.re Agrario. 52 (25): 61-65. 

LOZZIA G.C., RIGAMONTI I.E., 1990 - Influenza dell'ambiente e delle tecniche agrocolturali sulla presenza dei 

fitoseidi (Acarina: Phytoseiidae) in alcuni vigneti del Nord Italia. Atti Giornate Fitopatologiche 1990, 1: 449- 

458. 


16

LOZZIA G.C., RIGAMONTI I.E., 1994 - Modello di gestione integrata dell'agroecosistema vigneto. Atti Convegno 

"La difesa integrata dell'uva da tavola e da vino per gli aiuti comunitari del regolamento CEE 2078/92", Latina, 

26 XI 1994: 75-98. 

LOZZIA G.C., RIGAMONTI I.E., 1998 – Effects of weed management on phytoseiid populations in vineyards of 

Lombardy (Italy). Boll. Zool. Agr. Bachic., Ser II, 30: 69-78. 

REMUND U., NIGGLI U., BOLLER E.F., 1989 - Faunistische und botanische Erhebungen in einem Rebberg der 

Ostschweiz. Einfluss der Unterwichsbewirtschaftung auf das Ökosystem Rebberg. Landwirtschaft. Schweiz. 

Band. 2: 393-408. 

REMUND U., GUT D., BOLLER E.F., 1992 – Beziehungen zwischen Begleitflora und Arthropodenfauna in 

ostschweizer Rebbergen. Einfluss der botanischen Vielfalt auf okologische Gleichgewichte. Schweiz. Zeit. 

Obst- Weinbau, 128 (20): 528-540. 

REMUND U., BOLLER E.F., 1992 - Blühende Rebberge in der Ostschweiz: 3. Ergänzende Pollenfrassversuche mit 

Raubmilben. Schweiz. Zeit. Obst- Weinbau, 128: 237-240. 

VIDANO C., 1958 - Le cicaline italiane della vite (Hemiptera Typhlocybidae). Boll. Zool. agr. Bachic., Ser. 11, 1: 61- 

115. 

VIDANO C., ARNÒ C., ALMA A., 1988 - On the Empoasca vitis intervention threshold on vine (Rhynchota 

Auchenorrhyncha). In Vidano C. & Arzone A. (ed.), Proc. 6th Auchenorrhyncha Meeting. CNR-IPRA, Torino: 

517-524. 

WIEDMER U., BOLLER E.F., 1990 – Blühende Rebberge in der Ostschweiz: 2. Zum Pollenangebot auf den 

Rebenblättern. Schweiz. Zeit. Obst- Weinbau, 126: 426-431. 

ZANGHERI S., DONADINI P., 1980 - Comparsa nel Veneto di un Omottero neartico: Metcalfa pruinosa Say 

(Homoptera, Flatidae). Redia, 63: 301-305 


17

BIOPESTICIDES AND THEIR REGISTRATION IN THE UNITED STATE 

Sheryl K. Reilly 

Chief, Biochemical Pesticides Branch 

Biopesticides and Pollution Prevention Division 

Office of Pesticides Programs 

U.S. Environmental Protection Agency 

Biopesticides play an important role in organic crop production. Their proper use can significantly enhance the quality 

and production of crops targeted to address specific “niche market” needs. Biopesticides are regulated by the U.S. 

Environmental Protection Agency (USEPA) as pesticides and the cost of commercial development can be significant. 

Of the two broad classes of pesticides currently recognized by the USEPA, namely conventional chemicals and 

biological pesticides (or biopesticides), a substantial number belong to the biopesticide class, specifically the 

biochemical category. By definition, a biochemical pesticide is classified as such if it can be shown to possess a nontoxic 

mode of action on the target pest, and if it is naturally occurring or structurally similar and functionally identical to 

a naturally occurring substance. Biochemical pesticides are generally active at low concentrations and fairly specific in 

terms of their effects on a pest. USEPA is required by law to assure that any pesticide use in commerce will not result 

in unreasonable adverse effects to humans or the environment. In order to make this assessment, data requirements 

(e.g., mammalian toxicology, product identification and analytical methods, non-target plant and animal toxicology, 

fate, etc.) have been established for pesticide product registration. Registrants must address each data requirement, 

either by submitting a valid study or a request for a waiver of those requirements, prior to receiving formal product 

registration by the USEPA. Waivers of required data are based on scientific rationale or information that is known in 

the publically available, peer-reviewed scientific literature. For biochemical pesticides, the acceptability of data and/or 

waivers, as well as any proposed label uses, are reviewed by the Biopesticides and Pollution Prevention Division 

(BPPD) in the Office of Pesticide Programs of the USEPA. This division of the USEPA was formed in 1994 to facilitate 

the development of biopesticide products which are important tools in an integrated pest management program and 

offer alternatives to conventional chemical pesticides. Given the time and expense associated with product 

development, commercialization, and registration, registrants should work closely with the USEPA, grower groups, 

university and extension personnel, trade group(s) and others to ensure a successful product development process. 


18

DIFESA DELLE COLTURE DAI PATOGENI FUNGINI IN AGRICOLTURA BIOLOGICA: PROBLEMI, 

MEZZI DISPONIBILI E PROSPETTIVE 

Maria Lodovica Gullino, Angelo Garibaldi 

DI.VA.P.R.A. – Patologia vegetale, Università di Torino 

La difesa delle colture dall’attacco dei numerosi patogeni fungini da sempre rappresenta un problema di non facile ed 

immediata soluzione in molti sistemi colturali. Ciò è tanto più vero nel caso delle produzioni biologiche. 

La situazione può essere più o meno complessa nel caso di sistemi colturali differenti: ad esempio è attualmente 

possibilmente (ma non lo sarà forse più domani) proteggere la vite dagli attacchi di patogeni fungini mentre più 

complessa è la situazione per il riso o per le colture ornamentali. 

In questa relazione vengono pertanto presi in considerazione i principali problemi causati da patogeni fungini in alcuni 

sistemi colturali, evidenziando le difficoltà che si incontrano o che si possono incontrare nel loro contenimento. Si 

esaminano i mezzi di difesa disponibili, valutando, ove possibile, criticamente le reali possibilità di impiego, 

sottolineando l’esigenza, anche per il comparto dell’agricoltura biologica, che tutti i prodotti ammessi passino il vaglio 

di una idonea valutazione, a tutela del produttore e del consumatore. 

Viene ribadito il concetto che, in modo particolare nel caso delle produzioni biologiche, la difesa delle colture ini zia con 

l’adozione di tecniche colturali idonee e con l’impiego di varietà resistenti o scarsamente suscettibili nei confronti dei 

più importanti parassiti. 

Vengono forniti alcuni esempi relativi a colture di importanza economica in Italia e si discutono criticamente le 

prospettive future. 


19

TOXICITY AND ANALYSIS OF MYCOTOXINS AND THEIR OCCURRENCE IN SOME ORGANIC 

PRODUCTS 

M. Solfrizzo, M. Pascale, A. Visconti, G. Avantaggiato 

CNR, Institute of Science of Food Production, Viale Einaudi 51, 70125 Bari, I taly 

Fumonisins, ochratoxin A, and deoxynivalenol are among the most important mycotoxins due to their toxicity and 

frequent occurrence in foodstuffs, including cereals. Fumonisins are mainly produced by Fusarium verticillioides e F. 

proliferatum on maize before and soon after harvest. These toxins can cause equine leukoencephalomalacia, porcine 

pulmonary oedema, liver and kidney cancer in rodents, nephrotoxicity and immunosuppression. The occurrence of 

ochratoxin A in cereals depends on geographic areas and is related to the presence of Penicillium verrucosum and 

Aspergillus ochraceus. Ochratoxin A has a strong nephrotoxicity and, at higher dosage, is carcinogenic on renal 

proximal tubule. Deoxynivalenol occurs in wheat, barley, oats, rye, spelt and maize and less frequently in rice, 

sorghum, and triticale. It is produced by F. graminearum e F. culmorum two important plant pathogens. Recent 

toxicological evaluation by Joint FAO/WHO Expert Committee on Food Additives have established provisional 

maximum tolerable daily intakes of 2 e 1 µg/kg of body weight for fumonisins and deoxynivalenol, respectively. The 

provisional tolerable weekly intake of ochratoxin A of 0.1 µg/kg of body weight has been confirmed. 

For the analysis of cereal based food aimed to assess mycotoxin intake the use of validated analytical methods is 

essential. At this regard the European Committee for Standardization (CEN) has established method acceptability 

criteria for each mycotoxin. Recently, we have developed and validated by a collaborative study a new HPLC method 

for the determination of fumonisins in maize and maize based foods which has been adopted by CEN as European 

Standard Method. Official standard methods based on HPLC are available for the analysis of ochratoxin A in cereals, 

whereas a suitable method that respond to CEN criteria needs to be produced for deoxynivalenol. 

The consumption of organic products in Europe has increased in the last years but few information are available on their 

mycotoxin contamination as compared to conventional products. It is well known that agricultural practices play a role 

on mycotoxin formation in cereals. 

A total of 461 cereal samples collected in Italy, including 186 samples originating from organic production (136 soft 

wheat and 50 spelt) harvested in 1998-2000 and 275 samples from conventional production (138 soft wheat and 137 

durum wheat) harvested in 1999-2000 have been analysed for deoxynivalenol in our laboratory. A higher incidence of 

positive samples was found in organic wheat (65-100% of positive samples) and organic spelt (100% of positive 

samples) as compared to conventional wheat (38-80% of positive samples), whereas the highest levels of 

deoxynivalenol were found in conventional wheat (up to 6465 µg/kg). In particular, mean levels of deoxynivalenol in 

positive samples of organic soft wheat, organic spelt, conventional soft wheat and conventional durum wheat were 70- 

180 µg/kg, 148-192 µg/kg, 219-604 µg/kg e 251-1097 µg/kg, respectively. 


20

THE FUNGAL PHYTOTOXINS AND THEIR POTENTIAL ROLE AS HERBICIDES IN INTEGRATED 

CONTROL METHODS AGAINST WEEDS. 

Antonio Evidente 

Dipartimento di Scienze del Suolo, della Pianta e dell’Ambiente, Università di Napoli Federico II, Via Università 100, 

80055 Portici 

The man weed battle started long time ago due to the great hazard resulted from their wide diffusion in 

agricultural crops and pastures. Weeds have created heavy problems in reforestation activities and in important 

economical infrastructures. In the last 50 years, beside the traditional agrarian practises, the most diffused methods in 

controlling weeds are the use of chemical herbicides which are responsible for major environmental pollution with 

human and animal health risks. 

Therefore, numerous studies have been carried out to develop biological control using natural weed enemies as 

insects, viruses, bacteria and fungi but a particular interest has been directed towards phytopathogenic fungi which are 

the most common plant pathogens that in respect to other microorganisms may be applied with safety and simplicity. 

Phytopathogenic fungi are responsible, together with other microbial agents, of severe damages to important 

agrarian forestall and ornamental plants Frequently, these fungal species produce phytotoxins, bioactive metabolites 

with different chemical structure, mode of action, host specificity, biological activity and environmental impact. Toxins 

are generally substances which interfere with plant metabolism of which many are directly responsible of pathogenesis 

while others are not considered toxins at all 1,2 . 

Studies conducted in the last years have concentrated on controlling weed diffusion in important agrarian crops. 

Fungal pathogens isolated from some infected tissue of weeds are generally belonging to important taxonomic genera 

(Alternaria, Ascochyta, Drechslera, Fusarium, Melanconis, Phoma, Pyrenophora ). Some phytotoxins were isolated and 

characterised to determine their role in developing new methods for weed control, Their direct application or that of 

their derivatives or/and analogues with the pathogen may increase the herbicidal activity so improving the efficacy in 

terms of pathogenesis, virulence and herbicidal selectivity 

Structure elucidation and stereochemistry of phytotoxins may allow to plan their total enantioselective 

synthesis in order to obtain amounts suitable to carry out experiments in green houses and in fields to develop strategies 

for the biological integrated management of weeds. Some toxins may be derivatised to modulate their biological activity 

in terms of phytotoxicity and selectivity and to obtain new natural safe herbicides. 

Furthermore, phytotoxins can be used to develop a method for their analysis in complex samples. Therefore, if 

the phytotoxins are virulence features they may be used as biomarkers for in vitro selection of the best fungal strain to 

be used in the integrated management methods. In this communication, a brief overview will be presented on the results 

obtained on developing strategies to biologically control of Chenopodium album, Striga hermonthica and Orobanche 

ramosa, infesting many important agrarian crops like maize, beet root, millet, sorghum, sunflower, tomato, legumes, 

etc 2,3 . 

References 

1. Ballio, A.; Graniti, A. Experientia, 1991, 47, 751-826. 

2. Evidente, A.; Motta, A Phytotoxins from fungi, pathogenic for agrarian, forestal and weedy plants. In Bioactive 

Compounds from Natural Sources, Tringali, C. (Ed.), Taylor & Francis, London, 2001. Chapter 12, pp. 473-525. 

3. Evidente, A.; Abouzeid, M.A. Characterization of Phytotoxins from Phytopathogenic Fungi and their Potential Use 

as Herbicides in Integrated Crop Management. In Handbook of Sustainable Weed Management, Harminder, P.S. 

(Ed.), The Haworth Press Inc., Binghamton, NY, USA (2002) in press. 


21

ORAL COMMUNICATIONS 

• E. Anklam European Commission JRC, Geel, Belgium 23 

• C. Tuberoso Università di Cagliari 24 

• T. Pellicanò Università degli Studi di Messina 25 

• M. Kelderer Centro Sperimentale Laimburg, Bolzano 30 

• M. Rubbiani Istituto Superiore di Sanità, Roma 31 

• A. Ragni BioTecnologie BT (Italy) 32 

• P. Restani Università degli Studi di Milano 38 

• A. Santomauro Università di Bari 39 

• A. Di Muccio Istituto Superiore di Sanità 

• M. Benuzzi ASSOMETAB 41 

• G. Imbroglini MiPAF 42 


22

NEED FOR RESEARCH AND APPROPRIATE ANALYTICAL TOOLS FOR ORGANIC FOOD CONTROL 

Yona Siderer a, and Elke Anklam b 

a Interdisciplinary Centre for Technological Analysis and Forecasting, at Tel Aviv University, Ramat -Aviv, Israel 

b European Commission, Joint Research Centre, Food Products Unit, B -2440 Geel, Belgium 

Organic farming and the organic food market are growing fast. Due to health concerns, environmental consciousness, 

social status considerations and other reasons, consumers are interested in the products of organic farming. At the same 

time, they want more information about these products. 

Inspection procedures for confirming the origin of products seem to be insufficient and need to be supported by 

analytical tools. Not only should the products purchased on the market be checked for their authenticity but also 

products used during the various production steps (e.g. fertilisers, soil, animal feed, pesticides). Analytical research 

work should accompany the expansion of organic farming and organic food production. This should take into 

consideration different climate conditions, the variety of soils and soil constituents, the variety of plants and plants 

strains, and husbandry of livestock related to animal welfare. At the same time, the products (vegetables, fruits, milk, 

meat, and processed food) should be studied for their own qualities. 

Research into agricultural crops with various cultivation methods and the resulting natural or processed food is complex 

and lengthy. It is very difficult to compare the contents of nutrients and vitamins, as the constituents of such substances 

depend heavily on storage time, conditions and technology. Fast and general answers cannot be given on methods for 

verifying authenticity and safety. Attention should be focused on the planning of experiments, taking into consideration 

many variable parameters like soil quality, climatic and geographical conditions and crop variety. It is important to 

study a large number of samples, to be able to deduce meaningful conclusions from the results of the research. The 

research protocol should involve a large enough number of research participants as well. The results of such a 

combined study could then be used as analytical tools for assessing organic products. Later on, a farmer or a retail outlet 

would be able to add to the label that the product was checked and, e.g., found “clean” of pesticides, or that mycotoxins 

were found to be below legal levels after harvesting. These tests would be beneficial for interested customers and for 

the organic market sustainability. It should be emphasised that this approach of collaborative structured studies is 

preferable to the work of a single researcher wanting to study a particular question of her or his interest (or the 

stakeholder’s interest). 

Once analytical tools have been developed, firstly for a small number of products, but in higher numbers along the 

years, then legislation on organic food products might be changed to include a call for analytical tests to support the 

inspection system. 


23

METODO MULTIRESIDUO PER LA DETERMINAZIONE DI RESIDUI DI ESTRATTI VEGETALI IN 

ALIMENTI BIOLOGICI 

C.I.G. Tuberoso 

Dip. di Tossicologia Università di Cagliari 

Via Ospedale, 72 Cagliari 

Nell'agricoltura biologica si effettua il controllo dei pesticidi di sintesi, in quanto la normativa 2092/91 non ne consente 

l'uso, per cui la sola presenza di residui a livelli > di 0,01 mg/kg rende gli alimenti non conformi. I pesticidi naturali per 

i quali è consentito l'uso invece, di norma, non vengono controllati, principalmente per carenza di metodiche analitiche 

rapide, semplici e con buona sensibilità. Anche a livello di ricerca gli studi su questi pesticidi naturali sono molto 

limitati, per cui la conoscenza del loro livello di residui negli alimenti è quasi completamente sconosciuta. Gli insetticidi 

estratti da piante più utilizzati sono quelli ottenuti dalla Azadirachta indica, Derris elliptica, Ryania speciosa, e 

Chrysanthemum cinerariaefolium i cui principi attivi sono rispettivamente: azadirachtin, rotenone, rianodina con 

deidrorianodina e piretrina I e II. 

Il presente lavoro propone una metodica che consente la determinazione di questi antiparassitari naturali in maniera 

semplice e sensibile. La tecnica utilizzata è la cromatografia liquida accoppiata alla massa (HPLC-MS) 

Inizialmente si è ottenuta la separazione cromatografica dei principi attivi utilizzando una colonna C18, un gradiente di 

H2O e CH3CN al flusso di 0,2 ml/min ed un rivelatore a serie di diodi settato alle lunghezze d’onda ottimali per la 

determinazione dei singoli principi attivi. Nella determinazione col detector di massa è stata impiegata una sorgente 

APCI e tutti gli antiparassitari sono stati analizzati in SIM con due metodi (differenti per le temperature del probe, CDL 

e Block) che permettono di ottimizzare la risposta per la deidrorianodina, la rianodina e l’azadiractina (metodo 1) e per 

il rotenone e le piretrine (metodo 2). 

L’estrazione dei principi attivi da matrici vegetali è stata effettuata con acetato di etile, solvente che si è dimostrato 

ottimale per il recupero quantitativo di tutti i principi attivi. L'estratto non subiva alcuna purificazione in quanto non 

presentava alcun interferente. Nelle determinazioni quantitative la soluzione madre veniva preparata in estratto di 

matrice non trattata per eliminare l'effetto matrice. 

I limiti di determinazione ottenuti sono compresi tra 2 e 10 ppb. Il rotenone è l’antiparassitario che fornisce la risposta 

migliore permettendo di rilevare fino a 2 ppb. 


24

PRESENCE OF OCHRATOXIN IN EXPERIMENTAL WINES RELATED TO PESTICIDES 

TREATMENTS EMPLOYED ON GRAPES 

R. Lo Curto*, F. Vilasi*, T. Pellicanò*, P. Munafò**, G.mo Dugo*, 

* Università degli Studi di Messina, Dip.to Chimica Organica e Biologica. 

** Centro Analisi & Servizi S. r. l. via U. La Malfa, 18 - 98051 Barcellona P. G., Messina, Italia. 

Introduction 

Mycotoxins are a class of highly toxic chemical compounds produced, under particularly environmental conditions, by 

several moulds developing in many foodstuffs. Their presence is depending upon several factors such as: fungal strains, 

climate and geographical conditions, cultivation techniques and foodstuffs conservation (1). Mycotoxins may occur in 

various vegetal products as cereals, dried fruits, coffee beans, cocoa and beverages as beer and wine (2-4). Among 

mycotoxins very important are the ochratoxins. This term indicates a group of metabolites, having showing similar 

chemical structure, produced by strains of the genus Aspergillus (A. ochraceus) and Penicillium (P. ferrucosum) (1,5,6). 

The most studied, both for its diffusion and toxicological importance is Ochratoxin A (OTA) R-N-[(5-chloro-3,4dihydro-8-hydroxy-3-methyl-1-oxo-1H-2-benzopyran-7-yl)carbonyl]-phenylalanine. 

It has an oral DL50 level of 20 

mg/kg in rats and pig (7). OTA is highly toxic and causes severe animal and human intoxications, for example “Porcine 

nephropathy” and “Balcan Endemic nephropathy” (8-10). OTA exhibits nephrotoxic and teratogen activites and 

moreover suppressive actions on immune system, causing a diminution of immune globulin level and of other humoral 

factors in mice and chicken and a reduction of cell immune response (10,11). 

Recently, more attention was focused on Ochratoxin A levels in commonly consumed foods, especially fruits and 

cereals (12,13) and in their fermentation products like beer (14-16) and wine (17-25). 

As far as wine is concerned, available data regarding the presence of OTA are very discordant, some Authors reported a 

high toxin concentration (up to 7.0 ng\ml) with considerable level of contamination (incidence up to 92%) in red wine 

produced in southern regions of Europe and in North Africa. Other Authors reported OTA contamination levels ranging 

3.9% for white wines to 16.6% for red wines, even if these data were obtained by different analytical methods. 

Generally red wines contain a greater amount of OTA than white or rosè ones. These differences can be attributed to 

climatic factors, grape cultivation and storage conditions apart from wine-making techniques. The content of OTA in 23 

samples of red and white wine, produced in the year 2000 by three experimental vite, situated in three different Italian 

regions treated with different pesticides see tables 1,2,3), is reported in this work (26). 

Materials and methods 

Chemicals and Reagents 

Standard of OTA, sodium chloride, polyethylene glycol (Peg 8000), sodium hydrogencarbonate, glacial acetic acid and 

toluene were obtained from Fluka (Milano-Italy). Immunoaffinity columns were purchased from Vicam (Waterton, 

MA, USA).Acetonitrile, methanol and water (HPLC grade) were supplied from Carlo Erba Reagents (Milano-Italy). 

Wine samples 

A total of 23 wine samples (16 white 7 red samples), produced in the year 2000 by three experimental vite, situated in 

three countries “Avellino”-Campania, “Catania”-Sicily and “Grosseto”-Tuscany, were analysed. 

Wine characteristics and pesticide treatments 

White wines came from Sicily and Campania in the crop year 2000. Sicilian wines were produced from 15 -20 years old 

plants, grown up on Etna (300 m asl, S. Venerina, Catania, Italy), in a vulcanic soil. Vines were grafted with Inzolia and 

Carricante varieties in 1:1 ratio. Wines from Campania were produced from 25 years old plants grown up on 

Montefredane hills (700 m asl, Avellino, Italy), in a clayey soil. Vines were grafted with Fiano d’Avellino variety. Red 

wines from Tuscany were produced from 25 years old plants, cultivated in Maremma Toscana coast (100 m asl, 

Grosseto, Italy), on a calcareous soil. Vines were grafted with Sangiovese variety, Morellino clone. Vinification process 

was run within 24 hours after grape harvesting and it was run using the following scheme: 

White Vinification 

Newly – cropped Vitis vinifera grapes from Sicily and Campania were crushed destemmed and then soft pressed by a 

pneumatic press. Must was treated with SO2 (30 g/hl), pectolitic enzymes (1.5 g/hl) and vitamin C (5 g/hl) to favour 

clarification before fermentation; temperature was maintained at 8°C for 24 hours. Clear must was spiked with 20 g/hl 

of thiamine and ammonium phosphate as fermentation coadjuvans and 30 g/hl of selected yeasts and fermentation was 

run at 15°C. In order to remove lees, after fermentation wine was decanted into a tank and spiked with SO2 (5 g/hl). Ten 

days later wine was decanted again and treated with SO2 (5 g/hl). Wine was finally filtered twice trough 1 µ and 0.45µ 

cardboards filters, spiked with SO2 (2-3 g/hl), bottled in dark bottles and maintained at 4°C for the duration of the 

experimentation. Each grape sample was separately vinificated, following the above mentioned protocol. 

Red vinification 

Newly – cropped Vitis vinifera grapes from Tuscany were crushed and destemmed, then spiked with SO2 (5 

g/hl), added with selected yeasts (30 g/hl) and let to ferment for 10 days at 28°C, making three fullings a day. Wine was 

drawn from the vat and the vinasses pressed by a hydraulic press. After 4 weeks, lees were removed and the wine 


25

spiked with SO2 (2-3 g/hl). Wine was spiked again with SO2 (2-3 g/hl) and bottled in dark bottles at 4°C for the duration 

of the experimentation. 

Each grape sample was separately vinificated following the above mentioned protocol. All wines were stored in 

the dark at 4°C, and each sample was open immediately prior to analysis. 

Pesticide treatments. 

Pesticides were used at the doses recommended by the manufacturer and were sprayed with a manual sprayer in 

the three Italian vines. Each Sicilian samples but four, was subjected to a Sulfur treatment, followed by organic 

pesticide treatments, applied during grape ripening and repeated for six times every 15 days. Two samples were treated 

only with Sulfur (dry and wettable powder), one sample was treated only with organic pesticides (Dinocap – 

Penconazole) and one sample only with water in order to have a comparison with treated samples (table 1). Each 

sample from Campania but four, was subjected twice to Sulfur treatments every 10 days, followed by organic pesticide 

treatments applied during the vines maturation phase and repeated 6 times every 15 days. Two samples were treated 

only with Sulfur (dry and wettable powder), one sample was treated only with organic pesticides (Dinocap – 

Penconazole) and one sample only with water (table 2). Each Tuscan sample but four, was subjected twice to Sulfur 

treatments every 8 days, followed by organic pesticides tratments, applied during the vines maturation phase and 

repeated 9 times every 12 days. Two samples were treated only with Sulfur (dry and wettable powder), one sample was 

treated only with organic pesticides (Dinocap – Penconazole) and one sample only with water (table 3). 

Apparatus 

The analytical method proposed by Visconti et al. for OTA determination in wine was utilized. Utilizing commercial 

immunoaffinity columns and HPLC equipped whit a RF detector carried out Ochratoxin A determination.The 

chromatographic analysis was run by using a Shimadzu HPLC system equipped with a System Controller SCL-10 A, an 

RF-1AXL detector (λex=330 nm, λex=460nm), a LC 10A pump, a Rheodyne injector with a loop of 20 µl and a 

reversed-phase Supelco column C18 (15 cm x 4.6 mm, 5 µm particles) equipped with a guard filter (0.5 µm). Analyses 

were run at room temperature in isocratic conditions with a mobile phase composed of water \ acetonitrile \ acetic acid 

99:99:2 v\v\v at a flow rate of 1 ml\min. OTA quantification was made by measuring peak areas at OA retention time 

and by comparing them with calibration curve. Using a wine sample added of OTA made confirmation of OTA 

identification standard. HPLC chromatogram of a standard solution of Ocratoxin A is shows in Fig.1. The sensibility 

test of analytical method was of 0.01 ng\ml. 

Results and discussion 

Values of OTA found in wines are shown in tables 4 for Sicily and Tuscan respectively. OTA was not found in white 

wines produced in Campania while, of eight samples from Sicily, only six were found to be contaminated; the higher 

value found was 0.03 ng\ml in the sample treated with dinocap. The sample treated with quinoxifen and the treated with 

water were found not contaminated with OTA. Because of OTA concentration in this wine is very low, it is not possible 

to correlate its presence with pesticide treatments.Values of OTA found in red wines show that all samples are 

contaminated. The sample produced with grapes treated only with sulfur 80 PBWG, showed a Ochratoxin A content 

(2.00 ng\ml) higher than the other samples, followed by sample obtained from grapes treated with powdered sulfur 

(0.71 ng\ml). The concentration of OTA in the other samples spanned from 0.07 ng\mg in sample treated with 

“azoxystrobin” to 0.24 ng\mg in sample treated with “dinocap and penconazole”. The values of OTA found, are 

comparable with those reported in literature on micotoxins presence in red wines. 

The experimental viticulture located in Campania is not subject to pollution from OTA producing moulds. The 

experimental Sicilian cultivar seems is more easily contaminated with OTA since, of eight samples, six were found 

contaminated. 

Values of OTA found in red wines show that experimental vite located in Tuscany is strongly subject to infection from 

OTA producing fungi. Synthetic pesticides can reduce the OTA concentration from 96.5% in the sample treated with 

azoxystrobin, to 88% in the sample treated with dinocap and penconazole. Since OTA is strictly related to the growth of 

some toxigenic fungi on grapes, the different Ochratoxin A content in detected wine samples can be considered an 

efficiency test of the pesticides used. 

References 

1. Capuano, A., Dugo, G.nni, Restani P. (1999). Le micotossine. Tossicologia degli alimenti Ed. UTET, Torino, 

Italia. 

2. Blanc, M., Pittet, A., Munozbox, R., Viani, R. (1998). Behavior of ochratoxin A during green coffee roasting 

and soluble coffee manifacture. J. Agric. Food Chem.,46, 673-675. 

3. Solfrizzi, M., Avvantaggiato, G., Visconti, A. (1998). Use of various clean-up procedures for the analysis of 

ochratoxin A in cereals. J. Chromatogr. A, 815, 67-73. 

4. Pittet, A., Tornare, D., Huggett, A., Viani R. (1996). Liquid chromatographic determination of ochratoxin A in 

pure and adulterated solubile coffee using or immunoaffinity column clean-up procedure. J. Agric. Food 

Chem., 44, 3564-3569. 


26

5. Hohler, D. (1998). Ochratoxin A in food and feed: Occurrence, legislation and mode af action. Z. 

Ernaehrungswiss., 37, 2-12. 

6. Blank, R., Hohler, D., Wolffram, S. (1999). Ochratoxin in the food chain-Occurrence, toxicity, and 

decontamination. Uebers. Tierernaehr., 27, 123-163. 

7. Cerutti, G. I fattori tossici naturali (1992). Il rischio alimentare Ed. Tecniche Nuove, Milano, Italia. 

8. Creppy, E.E. (1998). Human ochratoxicosis and nephropathy in Egipt. Hum. Exp. Toxicol., 17, 124 -129. 

9. Castegnaro, M., Plestina, R., Dirheimer, G., Chernozemsky, I. N., Bartsch, H. (1991). Mycotoxins. Endemic 

Nephropathy and Urinary Tract Tumours, IARC Scientific Publication No. 115, International Agency for 

Research on Cancer, Lyon, France. 

10. Castegnaro, M., Mohr, U., Pfohl-Leszkowicz, A., Esteve, J., Steinmann, J., Tillmann, T., Michelon, J., 

Bartsch, H. (1998). Sex- and strain-specific induction of renal tumors by ochratoxin A in rats correlates with 

DNA adduction. Int. J. Cancer, 77, 70-75. 

11. Pfohl-Leszkowicz, A., Pinelli, E., Bartsch, H., Mohr, U., Castegnaro, M. (1998). Sex- and strain-specific 

expression of cytochrome P450s in ochratoxin A-induced genotoxicity and carcinogenicity in rats. Mol. 

Carginog., 23, 76-85. 

12. Scudamore, K. A., Nawaz, S., Hetmanski, M.T. (1998). Mycotoxins in ingredients of animal feeding stuffs: II. 

Determination of mycotoxins in maize and maize products, Food Addit. Contam., 15, 30-55. 

13. MacDonald, S., Wilson, P., Barnes, K., Damant, A., Massey, R., Mortby, E., Shepherd, M. J. (1999). 

Ochratoxin A in dried vine fruit: method developmentand survey. Food Addit. Contam., 16, 253-260. 

14. Degelmann, P., Becker, M., Herderich, M., Humpf, H.U. (1999). Determination of ochratoxin A in beer by 

high-performance liquid chromatography. Chromatographia, 49, 543-546. 

15. Nakajima, M., Tsubouchi, H., Miyabe, M. (1999). A survey of ochratoxin A and aflatoxins in domestic and 

imported beers in Japan by immunoaffinity and liquid chromatography. J. AOAC Int., 82, 897-902. 

16. Visconti, A., Pascale, M., Centonze, G. (2000). Detrmination of ochratoxin A in domestic and imported beers 

in Italy by immunoaffinity clean-up liquid chromatography. J. Chromatogr. A, 888, 321-326. 

17. Visconti, A., Pascale, M., Centonze, G. (1999). Determination of ochratoxin A in wine by means of 

immunoaffinity column clean-up and high-performance liquid chromatography. J. Chromatogr. A, 864, 89- 

101. 

18. Leitner, A., Zollner, P., Paolillo, A., Stroka, J., Papadopoulu-Bouraoui, A., Jaborek, S., Anklam, E., Lindner, 

W. (2002). Comparison of methods for the determination of ochratoxin A in wine. Analytica Chimica Acta, 

453, 33-41. 

19. Soleas, G. J., Yan, J., Goldberg, D.M. (2001). Assay ochratoxin A in wine and beer by high-pressure liquid 

chromatography photodiode array and gas chromatography mass selective detection. J. Agric. Food Chem., 

49, 2733-2740. 

20. Zimmerli, B., Dick, R. (1996). Ochratoxin A in table wine and grape juice: Occurrence and risk assessment. 

Food Addit. Contam., 13, 655-688. 

21. Majerus, P., Otteneder, H. (1996). Detection and occurrence of ochratoxin A in wines and grape juice. Dtsch. 

Lebensm.-Rundsch, 92,388-390. 

22. Burdaspal, P. A., Legarda, T. M. (1999). Ochratoxin A in wines and grape musts and juices produced in Spain 

and other European countries. Alimentaria, 299, 107-113. 

23. Otteneder, H., Majerus, P. (2000). Occurrence of Ochratoxin A (OTA) in wines: influence of the type wine and 

its geographical origin. Food Addit. Contam., 17, 793-798. 

24. Ospital, M., Cazabeil, J. M., Betbeder, A. M., Tricard, C., Creppy, E.E., Medina, B. (1998). Ochratoxin A in 

wines. Rev. Fr. Oenol., 169, 16-18. 

25. Festas, I., Herbert, P., Santos, L., Cabral, M., Barros, P. (2000). Alves, Ochratoxin A in some Portuguese 

wines: Method Validation and screening in Port wine and Vinho Verde Am. J. Enol. Vitic., 51, 50-154. 

26. R. Lo Curto, T. Pellicanò, F. Vilasi, P. Munafò, G.mo Dugo Food. Chem.(2003) (in press) 


27

Figure 1: HPLC chromatogram of standard OTA 

Table 1: Pesticide treatments on “Fiano di Avellino”(Campania) variety grapes 

Wine samples n° of pesticide treatments during grape ripening 

Sicily 1 1 Sulfur 80 Pb 6 Quinoxyfen250 SC 

Sicily 3 1 Sulfur 80 Pb 6 Fenarimol 12 SC 

Sicily 4 1 Sulfur 80 Pb 6 Azoxystrobin 250 SC 

Sicily 5 1 Dinocap 350 EC 6 Penconazole 100 EC 

Sicily 8 1 Sulfur 80 Pb 6 Sulfur 80 Pb 

Sicily 9 1 Sulfur 80 Pb 6 Dinocap350EC 

Sicily 10 1 Sulfur Powder 6 Sulfur Powder 

Sicily 11 1 Water 6 Water 

Table 2: Pesticide treatments on “Inzolia” and “Carricante”(Sicily) varieties grapes 

Wine samples n° of pesticide treatments during grape ripening 

Campania 1 2 Sulfur 80 Pb 6 Quinoxyfen 250 SC 

Campania 2 2 Sulfur 80 Pb 6 Fenarimol 12 SC 

Campania 3 2 Sulfur 80 Pb 6 Azoxystrobin 250 SC 

Campania 4 2 Sulfur 50 PS 6 Sulfur 50 PS 

Campania 5 2 Dinocap350EC 6 Penconazole 100 EC 

Campania 6 2 Sulfur 80 Pb 6 Dinocap 350 EC 

Campania 7 2 Water 6 Water 

Campania 8 2 Sulfur 80 Pb 6 Sulfur 80 Pb 


28

Table 3: Pesticide treatments on “Sangiovese” (Tuscany) variety grapes 

Wine Samples n° of pesticide treatments during grape ripening 

Tuscan 1 2 Sulfur 80 Pb 9 Quinoxyfen 250 SC 

Tuscan 2 2 Sulfur 80 Pb 9 Fenarimol 12 SC 

Tuscan 3 2 Sulfur 80 Pb 9 Azoxystrobin 250 SC 

Tuscan 4 2 Dinocap 350 EC 9 Penconazole 100 EC 

Tuscan 5 2 Sulfur Powder 9 Sulfur Polvere 

Tuscan 6 2 Sulfur 80 PBWG 9 Sulfur 80 PBWG 

Tuscan 7 2 Dinocap 350 EC 6+3 Sulfur + Quinoxyfen 250 SC 

Table 4: Concentration of OTA in wine samples nd: not detected 

Wine Samples Ochratoxin A (µg L -1 ) 

Sicily 1 n. d. 

Sicily 3 0.02 

Sicily 4 0.01 

Sicily 5 0.02 

Sicily 8 0.02 

Sicily 9 0.03 

Sicily 10 0.01 

Sicily 11 traces 

Tuscan 1 0.22 

Tuscan 2 0.11 

Tuscan 3 0.07 

Tuscan 4 0.24 

Tuscan 5 0.71 

Tuscan 6 2.00 

Tuscan 7 0.10 


29

EFFICACIA DELLA RYANIA SPECIOSA, COME INSETTICIDA NATURALE 

Markus Kelderer, Ellen Elias 

Centro Sperimentale Laimburg (BZ) 

Ryania speciosa (Vahl) è un arbusto appartenente alla famiglia delle Flacourtiacee presente nel clima tropicale del 

Centro- e Sudamerica. Le prime notizie di questa pianta risalgono al 1897 quando Cortes segnaló di aver trovato una 

pianta con un alcaloide tossico chiamato dagli indigeni ‚Matacucaracha‘. 

Negli anni 40 in agricoltura il legno macinato di Ryania speciosa venne usata contro diversi insetti nocivi. (es. in 

frutticoltura contro Cydia pomonella). Gli allegati 2b del Regolamento sull’agricoltura biologica 2092/91 inizialmente 

citavano preparati a base di Ryania speciosa come compatibili con i principi dell’agricoltura biologica. Con il Reg. 

1488/97 la Ryania speciosa venne tolta da tale lista poiché non era registrata come fitofarmaco in nessun paese della 

Comunitá Europea. In mancanza di alternative nel contenimento di alcuni tortricidi cardinali in frutticoltura biologica su 

richiesta delle associazioni dei produttori, l’argomento venne inserito tra i temi prioritari da affrontare nell’ambito dei 

progetti finalizzati ‚Difesa in agricoltura biologica‘. I lavori svolti nell’ambito dei 3 anni di ricerca sono: 

1) Raccolta bibliografica delle conoscenze finora presenti 

2) Sviluppo di un metodo di estrazione capace di estrarre il quantitativo maggiore di Ryanodina componente 

principale degli alcaloidi presenti nel legno di Ryania speciosa. A tale proposito si saggiarono diversi solventi 

(acqua, metanolo, etanolo, diclormetano, acetone, ecc.), diversi metodi di estrazione (Soxhlet, bollitura a riflusso, 

Ultraturrax, Estrazione con SFE, ) e diverse metodologie per determinare la Ryanodina (cromatografia su strato 

sottile TLC e HPLC). 

3) Biotest in laboratorio su diversi lepidotteri carpofagi (Cydia pomonella, Lobesia botrana, Pandenmis heperana, 

Adoxophyes orana, Mamestra brassicae) confrontando legno macinato di Ryania speciosa, Ryanodina pura ed 

estratti standardizzati. Vennero fatte sia prove contro le uova, le larve e gli adulti dei lepidotteri citati 

determinandone la DL50. 

4) Ricerca di sinergisti (Piperonilbutossido, ecc. ) per esaltare l’efficacia della Ryanodina come principio attivo. 

5) L’efficacia in pieno campo di diverse formulazioni a base di Ryania contro diversi fitofagi. 

6) Determinazione della persistenza del principio attivo in condizioni di pieno campo. 

7) Determinazione dei metodi analitici per il riscontro dei residui. 

Risultati: 

Tra i metodi di estrazione si è dimostrato particolarmente efficacie e di facile esecuzione il metodo Soxhlet usando 

come solvente il metanolo; la determinazone della Ryanodina si è effettuata con HPLC/UV-VIS con colonna RP-18. 

In laboratorio gli estratti di Ryania Speciosa non hanno dimostrato di avere un efficacia contro adulti ne contro uova dei 

citati fitofagi. L’efficacia contro le larve si è dimostrata variabile in funzione della specie. L’aggiunta di sinergisti ha 

migliorato l’efficacia degli estratti. Tale aumento dell‘efficacia varia a seconda della specie in oggetto. Le prove di 

campo confermano i risultati riscontrati in laboratorio. 


30

BIOPESTICIDES: EVALUATION PROCESS FOLLOWING THE APPLICATION 

OF THE DIRECTIVE 91/414/CE 

M. Rubbiani 

Istituto Superiore di Sanità 

Laboratorio di Tossicologia Applicata 

Viale Regina Elena 299 - 00161 Roma - ITALIA 

E mail: rubbiani@iss.it 

Directive 91/414/CE for new and existing (revision) biopesticides requests the same kind of full evaluation, in terms of 

risk assessment, provided for chemicals. 

The first application of the guidelines immediately showed the obvious differences between the two types of products 

(chemical and micro-organism based pesticides), and the need to distinguish within the critical end points addressed to 

both of them. 

This yielded to revise the data requirements for risk assessment (Annex II part B and Annex III part B of the Directive), 

because of the foreseeable problems due to the specific nature of the micro-organisms based pesticides. 

The different aspects regarding the critical end points of the evaluation process, such as mode of action, 

characterisation, toxicological and microbiological features, as well as the possible risk for human and environment are 

fairly highlighted. 

Moreover, the problems raised in relation to the use and handling of these products, as well as the possibility to develop 

new strategies for a more suitable toxicological and ecotoxicological evaluation (correctly hazard- and risk- based) are 

reported. 

The activity within Directive 91/414/CE evaluation procedures at EU level and the activity of the National Committee 

for the registration of biopesticides are also shortly described. 


31

ENTOMOPATHOGENIC NEMATODES IN BIOLOGICAL CONTROL: REALITY AND PROSPECTIVES 

M. Ricci and A. Ragni 

BioTecnologie BT srl Pantalla di Todi 06050 Perugia Italy 

Tel: ++39 – 075 - 895091 Fax: ++39 – 075 – 888 776 E-mail: mricci.bt@parco3a.org, aragni@parco3a.org 

General description 

Entomopathogenic nematodes (EPNs) are small (about 1 mm in length) round worms and are obliged parasite of 

insects. In the last few decades, they have been studied to develop new bio control agents (BCAs). 

EPNs belong to the phylum Nematoda, class Secernentea, order Rhabditida, sub-order Rhabditina, super-family 

Rhabditoidea, families Steinernematidae and Heterorhabditidae. So far, about 35 species have been described, but every 

year new ones are added. 

Bio-insecticide products based on EPNs consist of the infective juveniles (IJs) which represents a stage of their life 

cycle that can survive out of the insect bodies without feeding, relying on their fat reserves. IJs can survive in the soil 

for long periods (if the soil is wet they can survive for months) until locate a suitable insect host (Gaugler, 1988). 

Mode of Action and Life Cycle 

In the soil, the IJ searches for and infects a suitable insect host penetrating into its haemocoel via natural body openings 

(mouth, anus, spiracles). Once in the haemocoel, the IJs release a symbiotic bacterium (Photorhabdus or Xenorhabdus 

spp.) into the haemolymph. The bacteria rapidly multiply, killing the insect by septicaemia within 24-48 hours. The 

nematodes feed upon the bacteria and degraded insect tissue and develop to first generation adult males and females (in 

the Steinernematidae family) or hermaphrodites (in the Heterorhabditidae family). In both cases, after the eggs have 

been fertilized, they are laid in the insect body or remain in the mother body. The hatched larvae complete their cycle 

passing thorough 5 stages. EPNs can complete 1 to 3 generations, depending on the host insect size. As the insect 

resource becomes exhausted, most of the juveniles differentiate into a particular third stage that becomes the survival 

form of the life cycle (Infective Juvenile). The insect cuticle then ruptures and the IJs escape into the surrounding 

environment (Wouts 1980; Poinar 1990). 

Behaviour 

Because of their potential as biological control agents for insect pests, a great deal of research has been conducted on 

their behaviour and ecology, in particular the behavioural ecology of host-finding. Host-finding typically involves five 

steps; (I) host habitat selection, (II) localized search, (III) host orientation, (IV) host acceptance (or attachment), and (V) 

host suitability (Vinson, 1975). Individual species adopt different approaches to each of these steps. Host searching 

strategies can be divided into two approaches. Ambushing, where the environment moves past a waiting animal, is most 

efficient against high densities of highly mobile hosts. Cruising, where the animal moves through the environment, is 

most efficient against sedentary and widely distributed hosts (Huey and Pianka, 1981). The entomopathogenic 

nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora appear to occupy similar habitats and use 

similar hosts, yet they exhibit different searching behaviours. S. carpocapsae appears to be an ambusher and H. 

bacteriophora appears to be a cruiser. For these two different strategies energy reserves are potentially the most 

important endogenous regulatory factor. Nematode infective stages are non-feeding and rely on stored lipids as the 

principal energy reserve. S. carpocapsae is on average 30% lipid by dry weight (Womersley, 1990). The rate of decline 

in lipid levels may be correlated with their foraging activity. Thus, many parasite infective stages remain inactive unless 

stimulated. S. carpocapsae becomes inactive without stimulation while H. bacteriophora remains active (Gaugler and 

Campbell, 1991). Increased age, and the subsequent depletion of lipids, decreased the pathogenicity of S. carpocapsae 

and H. bacteriophora when the host was farther away (Vanninen, 1990). 

Symbiotic bacteria 

Symbiosis 

Entomopathogenic nematodes are symbiotically associated with specific bacteria. Steinernematidae have their 

mutualistic relationship with bacteria of the genus Xenorhabdus and Heterorhabiditae are associated with bacteria of the 

genus Photorhabdus. The two bacteria genera are gram-negative, belonging to the family of Enterobacteriaceae. 


32

Xenorhabdus spp. and Photorhabdus spp. are carried in the intestine of the infective stages of nematodes; Steinernema 

spp. have a special intestinal vesicle where the bacteria are kept while Heterorhabditis spp. have their symbionts in the 

lumen of the anterior part of the gut. 

Once the nematodes have penetrated a host, they release the bacteria symbiont in the haemolymph causing a 

septicaemia and the host dies. Inside the larval cadavers the bacteria growth and nematodes develop to adult stages, 

multiply and sexually reproduce. Although the nematodes can grow axenically (without any micro-organism) or on 

other bacteria, their reproduction is optimal only in presence of their natural symbiont. Thus, Xenorhabdus spp. and 

Photorhabdus spp. have the status of obligate symbiont because they provide essential nutrients for the correct 

multiplication of the nematodes (Boemare et al., 1997). 

The nematode-bacterium complex represents an exciting example of co-evolution that involves the interaction between 

the host and the nematode-bacterium complex, the relationship within the two symbiotic partners and the interaction 

between the bacterium and its bacteriocines. To investigate these three connections, it is necessary to study the insect 

defence reactions, the pathogenicity of the nematodes and the bacterium pathogenicity, symbiotic properties and 

lysogeny. 

Insects have an immune system based on phagocytosis and encapsulation of exogenous agents. The recognition of 

foreign bodies is mediated by the production of some humoral factors (Boemare et al., 1997). Entomopahtogenic 

nematodes are able to depress or to escape the immune defence of insect larvae. This is obtained both with nonrecognition 

by insect humoral factors and by escaping the phagocytosis and encapsulation. The nematodes secrete an 

immune-depressive factor against insect immuno-proteins directed against the symbiotic bacteria. As been reported also 

that axenic nematodes produce toxin(s) able to cause paralysis and death of the host (Simoes, 1994). Symbiotic bacteria 

display also pathogenic action them-self: they produce toxins and secrete enzymatic complex (proteases, lipases and 

phospholipases) that help the establishment of septicaemia in the host. Thus both partners of the symbiotic complex 

collaborate to kill the host. It is also been reported that axenic S. glaseri or its sole symbiont X. poinarii, have no 

entomopathogenic action, but this was restored after re-association of both partners (Akhurst and Boemare, 1990). The 

observed lysogenity of the bacterial symbiont is also useful for the symbiosis relationship because it helps the 

symbionts to compete with closely related bacteria. 

Production of useful molecules 

Both Xenorhabdus and Photorhabdus spp. can be grown independently from they nematodes partners under standard 

laboratory conditions. In vitro, bacteria secrete several extracelluar products: proteases, chitinases, lipases, 

phospholipases, antibacterial and antimycotic substances. Those compounds are produced also in vivo and the enzymes 

digest the tissues of dead larvae in order to provide nutrients for both bacteria and nematodes. The antifungal and 

antibacterial products are used to preserve the cadavers from the colonisation of other microorganisms. Those active 

substances have also been tested for their use in biolgical control of phytopatogenic fungi (Ng and Webster, 1997). 

In vitro, X. nematophilus and X. bovienii produce aliphatic amides (Park and Paik, 2001) and dithiolpyrrolones tested 

for antineoplastic activity (Webster and Chen, 1999). 

Recently, strains of Photorhabdus capable to secrete exotoxins (Bowen et al., 1998), or to produce endotoxins (Ragni et 

al., 1997), with oral insecticidal activity, have been described. This new source of natural toxins could help both the 

fight against the development of insect resistance as well as novel sources of specific activities in combination with 

other known bio-pesticides. 

Target organisms 

Due to their poor resistance to dry conditions, EPNs are mainly applied against soil dwelling insects. Otiorrhynchus 

sulcatus F. (Coleoptera, Curculionidae) is considered one of the major worldwide insect pests. The larvae feed on the 

root system of several plant species (mainly ornamentals and nursery stock) producing its consequent decay and very 

significant economic losses. A part other Curculionids such as Conorhychus mendicus, Balaninus elephas, Diaprepes 

abbreviatus and Cylas formicarius on ornamentals, berries, citrus, sugar beet and chestnuts, other pests are: Scarabeids: 

Popilia japonica, Maladera matrida, Phyllopertha horticola , Aphodius spp. on lawns, orchards, nurseries and sweet 

potatoes; Diptera: Sciarids, Phorids, Scatopsids, Cecidomids on mushrooms, greenhouses; Lepidoptera: Noctuids on 

vegetable crops. Several studies have been performed to find out if EPNs can be used on the aerial part of the plants 

against chewing and sucking insect, but, yet, without any important success. 

Application Technology 

Particular strategies must be developed in order to insure the successful delivery of the nematodes to the target site and 

target insect. Many parameters must be investigated to improve EPNs performance. One of these is the determination of 

target insect life-stage susceptibility, since different life stages of different species are not equally susceptible. It has 

been shown that pest population levels and behaviour have a great influence on nematode performance and must be 

considered carefully. Often, larval stages of insects such as borers are not accessible to nematodes. Selecting the most 


33

appropriate nematode species and/or strain is important for efficacy and commercial development. Abiotic factors such 

as soil type, soil temperature and moisture, and biotic factors, including pathogens and predators, can greatly influence 

the nematodes ability to effectively kill the target pest. 

Application strategies, including field dosage, volume, irrigation and appropriate application methods, are very 

important, especially if nematodes are to be integrated with other control strategies. Compatibility with other 

agrochemicals has been demonstrated allowing the use of EPNs in existing Integrated Pest Management programs. Also 

crop morphology and plant phenology must be considered. Additional researches have shown the potential for 

entomopathogenic nematodes to be used in other habitats (e.g. aquatic, foliar, and cryptic), and in manure. 

EPNs are applied to the crops by common sprayers (also by drip irrigation); attentions should be taken in order to avoid 

sedimentation of nematodes by continuous agitation and to avoid exposure of nematodes to direct sunlight, preparing 

the suspension under a shade and applying the product at late afternoon. It is important to pre-irrigate the soil with at 

least 6 mm of water and an irrigation post application of about 6-12 mm of water. The treated area should be irrigated 

frequently (at least every 1-3 days with a minimum of 6 mm of water) to maintain appropriate soil moisture for EPNs. 

Production 

EPNs can be produced in vivo using insects (in general, the last instar larvae of the Lepidoptera Galleria mellonella). 

This method requires low technology but, in the other hand, requires a constant source of healthy insects and a large 

amount of manpower and thus costs are enormous, in particular, in western countries. EPNs can be produced in vitro, 

both in solid and in liquid media. The latter method is the most sophisticated because requires innovative know how and 

modern facilities and equipment; this method allows a strong reduction in the production process costs. 

Formulation 

Entomopathogenic nematode based-product requires a reliable and stable formulation. This has been a difficult task, 

because the larger markets are demanding a product with a minimum shelf life of six months when stored at room 

temperatures (20°-25°C). Nematode products contain living animals that have certain temperature, oxygen and moisture 

requirements necessary for their survival and effectiveness as control agents, cannot reach this market requirements. 

While no nematode formulation has been completely successful in reaching these goals, some have come very close 

(Georgis, 1992) such as a product based on the nematode Steinernema carpocapasae formulated in granules. A part this 

exception, due to the particular physiology of this species, the most common available formulations are based on clays 

with a limited shelf life (1-2 months) at 5-10°C. 

Perspectives 

To broaden the use of EPNs in Biological Control or IPM Programs, it is necessary to achieve positive results in the 

following fields: 

New Agricultural Targets; for instance: Grillotalpa grillotalpa, Melolontha melolontha, Leaf miners, Fruit flies 

(Bactrocera sp., Ceratitis sp., Ragoletis sp.), Aleurodids, Aphids. 

Market Needs; following the market needs, several aspects need to be improved. The reduction of production costs may 

be achieved by new technology and/or new strains with new activity, higher infectivity and higher production. The 

product shelf life can be extended by new technology and new strains capable to live in dry conditions. 

Extended applications; the following applications may extend the use of EPNs: foliar application, stock farm use (ticks, 

fleas, flies), domestic use (flies, cockroaches, fleas). 

Particular attention is dedicated to new entomopatogenic nematodes able to survive and parasitize hosts at extreme 

environmental conditions such as cold, warm and dry environments and to the exploitation of the biological active 

molecules produced by the symbiotic bacteria. Related to these topics, BioTecnologie B.T., a small Biotech company, 

based in Umbria Region (Italy), is developing two innovative products: a new strain of EPN active at very low 

temperatures and an innovative bio-insecticide based on endotoxins produced by a particular strain of the nematode 

symbiotic bacterium Photorhabdus luminescens: 

- Entomopathogenic nematode active at low temperature 

Many soil dwelling insect pests can or should be controlled when the temperatures are below 12°C (late autumn or early 

spring). The most important representative is Otiorhynchus sulcatus (Coleoptera, Curculionidae), a serious pest of soft 

fruits and ornamental plants. The inadequacies of chemical pesticides, the banning of the most persistent ones (e.g. 

aldrin) and the increasing dispersal of the insect, has led to an urgent need of a new and safer pest control method 

(Zimmermann, 1996). The available EPN based products, lack on effectiveness at temperatures below 12-15°C. 

A nematode strain, belonging to the species Steinernema kraussei, has been discovered and it is very active against O. 

sulcatus larvae even at 3°C (Ricci and Fridlender, 1999). The strain is also effective against larvae of B. elephas at 8°C. 


34

It can be reproduced in vivo at 15°C and in liquid culture at 20°C without loosing its cold activity. A product based on 

this nematode is actually under development. 

- Bio-insecticide based on endotoxins produced by a particular strain of the nematode symbiotic bacterium 

Photorhabdus luminescens 

It has been discovered (Ragni et al., 1997) a newly characterize strain of Photorhabdus luminescens, denominated 

XP01, which is highly toxic when directly administered by food contamination assays to neonates larvae of the 

Lepidopetran pests Mamestra brassicae and Cydia molesta. Further characterization made with Mamestra brassicae, as 

target insect, showed that the activity of this strain is caused by an intracellular compound that is not secreted into the 

culture media. In fact, the surnatant of fermented broth of XP01 had no toxic activity and this discovery was different 

from what was reported for the W-14 strain of P. luminescens (Bowen et al., 1998). It was demonstrated that the toxic 

effect of XP01 is maintained even when the bacterial cells are killed. The discovery of Photorhabdus luminescens strain 

XP01 indicates the existence of a presumably new family of entomotoxic proteins present in Photorhabdus and 

probably in Xenorhabdus spp. These insecticidal toxins could be used as an active bio-pesticide ingredient in a similar 

manner to the delta-endotoxins of B. thuringiensis. This new source of natural toxins could help both the fight against 

the development of insects resistance as well as serve as sources of novel specific activities in combination with other 

known biopesticides. 

Further studies could lead to understanding of the genetic control involved in the expression of XP01 insecticidal 

toxins. 

Conclusions: An Analysis of the Commercial Entomopathogenic Nematode Product 

In this type of analysis, commercial and scientific factors should be sorted into four categories either as ‘Strengths, 

‘Weakness’, ‘Opportunity’, or ‘Threat’ (source: Pest Management Resource Centre with the following information as 

available at the site: http://www.pestmanagement.co.uk/special/microb/com_prod.html). 

Product Strengths 

- Consistent supply available; supply will be consistent when sales forecasts is accurate and production levels are 

predictable. Most EPN species can be produced on a large scale, up to a capacity of 80,000 litre fermenters, at a 

concentration of 150-300,000 infective juveniles per millilitre. 

- The product is safe and ‘natural’; comprehensive data packages have been developed to support the safety claims 

made about EPN products. Workers benefit from not having to wear protective clothing during product application. 

Studies have also shown that EPN applications do not have a significant impact on non-target insects (Georgis et al., 

1991). 

- Products do not require registration; registration regulations vary from country to country and have not 

been standardized in Europe. In most countries EPN’s are regarded as animals and are exempt from pesticide 

regulations. This means that once efficacy data has been generated, products can be introduced. The commercial 

advantage of this is that product sales can begin at least three years faster than conventional products. 

- Products are easy to use; no additional equipment is required to apply EPN products, instructions have been 

designed to be similar to conventional pesticides making their use familiar to growers. 

Product Weaknesses 

- The insect life cycle; the susceptible stage of the insect life cycle may be in an inaccessible niche, or in a niche 

unsuitable for EPN survival. For example, excessively wet-dry soil or foliage. 

- Insect behaviour; insect behaviour or physiology may reduce EPN product efficacy. Gaugler (1988) suggested 

that the tendency of quiescent soil insects, especially pupae, to release carbon dioxide in bursts rather than 

continuously reduced the ability of infective juveniles to follow a chemical gradient to the host. It has been reported 

that when exposed to infective juveniles of Steinernema carpocapsae Weiser (All strain) American cockroaches, 

Peripaneta americana (L), actively groomed nematodes from legs and antennae to prevent infection. 

- Efficacy may be limited at hot and cold temperature; for example, control of black vine weevil Otiorhynchus 

sulcatus (F.) larvae in blackcurrant crops by Steinernema carpocapsae was limited at temperatures below 15 °C. 

- Product formulation; products should have a shelf life at room temperature. This can provide more flexibility in the 

distribution system. For example, a product shelf life at room temperature makes it easier to deliver and store the 

product in a warm climate, or the distributor could reduce costs by taking more products at one delivery and risk a 

temporary reduction in sales. Conversely, if the growers have cold storage facilities, as in the mushroom market, a 

sophisticated formulation may not be required, reducing development costs. 


35

- EPN products must be applied to moist soil; EPN’s can survive dry conditions (Womersley, 1990) but cannot search 

for and locate the insect host. 

Product Opportunities 

- The market value; it is important that the market supports growing sales of the newly introduced product at a 

price that immediately provides a return on the investment of the initial product development. 

- No chemical available / phytotoxic effects/ chemical not effective; For example, in American cranberry crops 

no conventional chemical is available to control the Cranberry Girdler Chrysoteuchia topiaria, or strawberry root 

weevil Otiorhynchus ovatus (L). Growers were limited to using cultural methods to prevent pest damage until an 

EPN product was developed. In mushroom crops application of insecticides reduced yields. 

- EPN searching behaviour; in field grown crops it is particularly difficult to apply insecticides at the point 

where the pest is causing damage. In strawberry plants grown under plastic access to the roots where black vine 

weevil larvae cause damage can be difficult. However, by applying EPN’s through T-Tape irrigation systems 

infective juveniles introduced below the soil surface can search for and infect larvae developing in the strawberry 

roots. 

- Knowledge of the pest life cycle; expert knowledge obtained from universities and extension services can be 

used to determine the correct time for product application. It is important that the insect biology is understood to 

prevent ‘mysterious’ product failures. 

Product Threats 

Grower confidence; any change in the product can cause problems with grower confidence. Even a change in the 

colour of the product container can cause suspicions that something is not quite right with the production system. 

Product education; in the initial stages of product introduction distributor and grower education is vital. If the 

growers misuse the product and observe damaged crops it is unlikely that they will continue to take risks on a ‘new’ 

idea. It is easy for a poor reputation to build up and almost impossible to recover confidence in the market. 

- Competitive conventional insecticides; the development of new conventional chemicals may address 

application problems, for example slow release formulations. 

References 

Akhurst, RJ and N. Boemare. 1990. Biology and taxonomy of Xenorhabus. In: Entomopathogenic nematodes in 

biological control R. Gaugler and H.K. Kaya, eds. CRC Press; Boca Raton FL. pp. 75-90. 

Boemare N., A. Givaudan, M. Brehélin and C. Laumond. 1997. Review article. Symbiosis and pathogenicity of 

nematode-bacterium complex. Symbiosis Vol. 22: 21-45 

Gaugler, R. (1988). Ecological considerations in the biological control of soil-inhabiting insects with entomopathogenic 

nematodes. Agriculture, Ecosystems and Environment 24 351-360. 

Bowen D.J., T.A. Rocheleau, M. Blackburn, O. Andreev, E. Golubeva, R. Bhartia, and R.H. ffrench-Constant. 1998. 

Insecticidal toxin from the bacterium Photorhabdus luminescens. Science. 280. 2129-2132 

Gaugler, R. and J. F. Campbell, 1991, Entomopathogenic nematode behavioural response to oxamyl, Ann. appl. Biol. 

119, p. 131-138 

Georgis, R., Kaya, H.K., and Gaugler, R. (1991). Effect of steinernematid and heterorhabditid nematodes (Rhabditida: 

Steinernematidae and Heterorhabditidae) on non-target arthropods. Environ. Entomol. 20:815-822. 

Georgis, R. 1992. Present and future prospects for entomopathogenic nematode products. Biocontrol Science and 

Technology 2: 83-99. 

Huey, R. B., and E. R. Pianka, 1981, Ecological consequences of foraging mode, Ecology 62, 991-999. 

Ng K.K. and Webster J.M.. 1997. Antimycotic activity of Xenorhabdus bovienii (Enterobacteriaceae) metabolites 

against Phyotophthora infestans on potato plants. Can. J. Plant Pathology 19 (2): 125-136. 

Park S.H. & Paik S.U. 2001. Novel aliphatic amide having anticancer property. PCT Application WO01/49656 

Poinar, G. O., Jr., 1990, Biology and Taxonomy of Steinernematidae and Heterorhabditidae, In Entomopathogenic 

Nematodes in Biological Control, R. Gaugler and H. K. Kaya Eds, CRC Press, Boca Raton. pp. 23-58 

Ragni A., Valentini F and Fridlender B.1997. “Insecticidal Bacteria”. PCTIL97/00246 

Ricci M. and Fridlender B., New entomopathogenic nematodes active at low temperatures. PCT/IL99/00218 

Simoes, N. 1994. Virulence factors produced by the entomopathogenic nematode Steinernema carpocapsae during 

parasitism. Proce. of the Vith Intern. Colloquium of Invertebrate Pathology Aug.28-Sept. 2. 1994. Montpellier, France 

p. 116-119 

Vanninen, I., 1990, Depletion of endogenous lipid reserves in Steinernema feltiae and Heterorhabditis bacteriophora 

and effect on infectivity, Proceedings and Abstracts of the 5th International Collogium on Invertebrate Pathology and 

Microbial Control, Adelaide. 


36

Vinson, S. B., 1975, Biochemical coevolution between parasitoids and their hosts, In Evolutionary Strategies of 

Parasitic Insects and Mites, P. W. Price Ed., Academic Press, New York. 

Webster J.M & G. Chen. 1999. Dithiolpyrrolones and their corresponding and dioxides as antineoplastic agents. PCT 

Application WO99/12543. 

Womersley, C. Z., Dehydration Survival and Anhdrobiotic Potential, 1980. In Entomopathogenic Nematodes in 

Biological Control, R. Gaugler and H. K. Kaya Eds, CRC Press, Boca Raton. 

Wouts, W.M., 1980. Biology, life cycle and redescription of Neoaplectana bibionis Bovien, 1937 (Nematoda: 

Steinernematidae). J. Nematology 12 1 62-72. 

Zimmermann, G., 1996. Microbial control of vine weevil. Proceedings of the second international workshop on vine 

weevil (Otiorhynchus sulcatus Fabr.) (Coleoptera, Curculionidae). Braunschweig, Germany, May 21-23, 1996. 


37

DETERMINAZIONE DEL CONTENUTO DI MICOTOSSINE IN PRODOTTI COMMERCIALI 

PROVENIENTI DA AGRICOLTURA TRADIZIONALE E BIOLOGICA 

B.Beretta, C.Ballabio, F.Tacchini, A.Cattaneo, C.L.Galli, C.Gigliotti*, P.Restani 

Dipartimento di Scienze Farmacologiche - Università degli Studi di Milano, via Balzaretti, 9 - 20133 Milano 

*Dipartimento di Produzione Vegetale - Università di Milano, via Celoria, 2 - 20133 Milano 

Le micotossine sono metaboliti secondari prodotti da alcuni funghi filamentosi che possono infestare le derrate 

alimentari. Questi composti sono stabili e permangono nell'alimento dopo la morte del micete produttore; sono resistenti 

ai lunghi periodi di conservazione ed ai comuni trattamenti industriali e casalinghi di preparazione dei cibi. 

Tra le micotossine più importanti dal punto di vista agro-economico e della salute pubblica si ritrovano le ocratossine e 

la patulina. 

La patulina è prodotta da alcune specie di Aspergillus e Penicillium che possono contaminare alcuni frutti e cereali ed in 

particolare la mela. E' una molecola relativamente instabile, ma è termoresistente se posta in ambienti acquosi e acidi. 

Diversi studi hanno portato a considerare tale composto come potenziale cancerogeno per l’uomo, anche se ulteriori 

approfondimenti sono necessari per comprendere se l’effetto cancerogeno possa manifestarsi alle concentrazioni 

normalmente riscontrate negli alimenti. 

Le ocratossine sono prodotte da alcune specie di Aspergillus e Fusarium e sono note per la loro nefrotosicità. Il 

composto più tossico presente in questo gruppo è l'Ocratossina A (OTA), una molecola abbastanza stabile che può 

passare inalterata attraverso la catena alimentare e ritrovarsi intatta in carne, cereali e derivati, 

L’oggetto di questo studio si articola nella valutazione: 

• della presenza di patulina in omogeneizzati e succhi di mela provenienti da diverse strategie agricole (coltivazione 

tradizionale, lotta integrata e agricoltura biologica); 

• della presenza di OTA in farine di cereali destinate alla prima infanzia, provenienti da diverse strategie agricole 

(coltivazione tradizionale, lotta integrata e agricoltura biologica); 

• della qualità tossicologica dei prodotti analizzati in funzione della diversa provenienza agricola. 

Per la ricerca della patulina sono stati analizzati 23 omogeneizzati di mela provenienti da agricoltura tradizionale, 

biologica e da lotta integrata e 21 succhi di mela provenienti da agricoltura tradizionale e biologica. 

Per la ricerca dell'OTA sono stati analizzati 30 lotti di semolino, 33 lotti di crema di riso, 26 lotti di crema di mais e 

tapioca e 30 lotti di crema multicereali. Anche questi prodotti derivavano da agricoltura tradizionale, biologica e da lotta 

integrata. 

Il protocollo di analisi prevede una prima fase di estrazione-purificazione del campione ed una seconda fase di analisi in 

HPLC. 

Tutti i campioni di omogeneizzati analizzati presentavano concentrazioni di patulina al di sotto del limite di legge 

stabilito in 50 µg/kg; la concentrazione maggiore riscontrata era di 6,39 µg/kg e 5,97 µg/kg per i prodotti da coltura 

tradizionale e da lotta integrata, rispettivamente. L'analisi statistica non ha evidenziato differenze significative tra valori 

determinati nei campioni provenienti dalle due strategie agricole considerate. 

Il più alto valore di patulina riscontrato nei succhi di mele provenienti da agricoltura tradizionale era 3,03 µg/kg, mentre 

i risultati relativi ai prodotti biologici erano estremamente variabili con un valore massimo di 28,24 µg/kg. Dal 

confronto dei dati, dal punto di vista statistico, si è evidenziata una differenza significativa tra le due tipologie di 

prodotti inclusi nello studio. 

Per quanto riguarda la ricerca dell'OTA, nessun campione di crema di mais e tapioca e nessun lotto dei prodotti 

provenienti da lotta integrata conteneva la micotossina in quantità rilevabili. Tra i lotti di semolino analizzati 

provenienti da coltivazione tradizionale, 2 dei 6 campioni risultati positivi superavano il valore limite fissato per legge 

in 0,5 µg/kg (0,58 e 0,65 µg/kg); mentre un solo lotto biologico presentava O,18 µg/kg di OTA. Tra le creme di riso 

incluse nello studio solo nei campioni biologici è stata rilevata la OTA ed in particolare due lotti superavano il limite di 

legge, con valori di 0,7 e 0,74 µg/kg. Le analisi delle creme multicereali hanno evidenziato la presenza di OTA solo nei 

prodotti da agricoltura tradizionale e nessun campione superava il limite di legge. 

Dai risultati ottenuti, il contenuto di OTA non sembra direttamente correlabile alla pratica agricola seguita; infatti nei 

semolini i valori più alti si sono riscontrati nei campioni provenienti da agricoltura tradizionale, mentre per le creme di 

riso si è verificato l’opposto. 

Le conclusioni di questo studio suggeriscono che la presenza di micotossine è un problema quanto mai attuale; i 

prodotti in commercio da noi analizzati, pur essendo nella quasi totalità dei casi nei limiti di legge, indicano la assoluta 

necessità di controllare le derrate alimentari e non trascurare i prodotti biologici che per le modalità di coltivazione e 

conservazione possono essere a maggior rischio di contaminazione. 

Beretta B., Gaiaschi A., Galli C.L., Restani P., 2000, Patulin in apple-based foods: occurrence and safety evaluation. 

Food Add. Contam., 17, 399-406. 

Beretta B., De Domenico R., Gaiaschi A., Ballabio C., Galli C.L., Restani P., 2002, Ochratoxin A in cereal-based baby 

food: occurrence and safety evaluation. Food Add. Contam., 19, 70-75. 


38

PROTECTION OF GRAPEVINE FROM POWDERY MILDEW BY USING NATURAL SUBSTANCES AND 

THE MICROBIAL ANTAGONIST AMPELOMYCES QUISQUALIS 

Agostino Santomauro (1) , Giuseppe Tauro (2) , Maurizio Sorrenti, Francesco Faretra (1) 

(1) Dipartimento Protezione delle Piante e Microbiologia Applicata, University of Bari 

(2) Centro di Ricerca e Sperimentazione in Agricoltura "Basile Caramia", Locorotondo (Bari) 

Powdery mildew, caused by Uncinula necator (Schw.) Burr., is one of the most common and severe diseases of 

grapevine, causing heavy yield losses wherever the crop is intensively grown. Many fungicides are available for its 

control. Recently, however, the rapid diffusion of organic agriculture has stressed out the lack of solid experimental 

data on the effectiveness of natural substances and microbial antagonists that are potential alternatives to the use of 

chemicals. 

The present paper reports the results of field trials carried out in 1998, 2000 and 2001 in order to evaluate the 

effectiveness of natural substances and the mycoparasite Ampelomyces quisqualis Ces. for the control of grapevine 

powdery mildew. 

The statistical scheme of randomized blocks with four replications and plots of 8-10 plants was adopted in all 

trials. The first two sprays were always carried out at the beginning and at the end of blossoming. Afterwards, 

treatments were carried out at one-week intervals until véraison. The tested spray schedules are listed below. 

Tested compounds Formulates Rates Year of employment 

(g or ml/ha) 1998 2000 2001 

Ampelomyces quisqualis + 

AQ 10 (Bio Intrachem) 

50 + • • • 

Mineral oil 

Ultra Fine Oil (Bio Intrachem) 2000 

Ampelomyces quisqualis + 

AQ 10 (Bio Intrachem) 

50 + 

• 

Pinolene 

Vapor-Gard (Bio Intrachem) 2000 

Sodium bicarbonate + 

RPH (Carlo Erba) 

5000 + • • • 

Mineral oil 

Ultra Fine Oil (Bio Intrachem) 2000 

Sodium bicarbonate + 

RPH (Carlo Erba) 

5000 + 

• 

Pinolene 

Vapor-Gard (Bio Intrachem) 2000 

Sodium bicarbonate RPH (Carlo Erba) 5000 • 

Foliar fertilizer Trym (Italpollina) 150 • 

Powder milk Vigor Latte (Mignini) 10000 • 

Mineral oil Ultra Fine Oil (Bio Intrachem) 10000 • 

Pinolene Nu - Film 17 (Chimiberg) 10000 • 

Pinolene Vapor-Gard (Bio Intrachem) 10000 • • 

Potassium salts of fatty acids MYX 403.2-050 (Mycogen) 10000 • 

Sulphur Zolfo WG (Bayer) 2500 • 

Sulphur Kumulus Tecno (Syngenta) 5000 • 

Symptom severity was assessed two to four times for each trial by observing 250-300 bunches per plot and 

counting infected berries. An empirical scale with 8 class of infection was used to calculate the following parameters: 

the percentage of infected organs (Tehon’s “prevalence”), the disease severity (Tehon’s “destructiveness”) and its 

weighted average value (according to the McKinney Index). All data were submitted to variance analysis and mean 

values were separated by Duncan’s Multiple Range Test. 

During 1998 and 2000, powdery mildew infections interested 77% and 65% of bunches in untreated plots, 

respectively. The values of McKinney Index, however, were not very high, reaching 17% and 13%, respectively. Under 

these conditions of medium disease pressure, all of the tested compounds showed to be effective against powdery 

mildew, decreasing significantly symptom severity on both berries and rachis, as compared to the untreated check. In 

particular, sodium bicarbonate (used either alone or in mixture with mineral oil) and pinolene showed the highest 

effectiveness, allowing reductions up to 90% of symptom severity. These results were substantially confirmed even 

under the higher disease pressure occurred in the trial carried out in 2001. In that year, powdery mildew symptoms were 

observed on 88% of bunches in untreated plots, with a value of McKinney Index as high as 34%. Even under such 

conditions, pinolene and sodium bicarbonate, used either alone or in mixture, allowed a significant reduction of the 

disease. Moreover, very interesting results were obtained with powder milk that yielded the best control of the disease. 

Repeated sprays with pinolene or potassium salts of fatty acids had some negative side effects on bunches, by 

removing waxes from the surface of berries and causing persistent bad smell. Repeated sprays with milk left visible 

residues, thus getting dirty bunches. Such effects represent a serious limitation for their usage on table grape, but should 

be negligible for wine grape. 

In conclusion, the tested natural substances and A. quisqualis showed an interesting, although not complete, 

effectiveness against grapevine powdery mildew. Yet, their exclusive usage in the protection of table grape seems can 


39

not be recommended due to their partial effectiveness and negative side effects. On the other hand, they might found a 

larger application in the protection of wine grape from powdery mildew in organic agriculture as well as in integrated 

disease management. However, further investigations are needed in order improve knowledge and rule out any 

detrimental effects on taste, flavor and other qualitative characteristics of wine. 

Acknowledgements: Work supported by the Italian Ministry of Agricultural and Forestry Policies in the frame of the 

research project “Technical tools for crop protection in organic agriculture” – Sub-project “Natural substances in crop 

protection from fungal diseases”. 


40

I MEZZI TECNICI PER L'AGRICOLTURA BIOLOGICA: UN SETTORE IN SVILUPPO SENZA LA CERTEZZA 

DELLA NORMATIVE CHE LO REGOLANO 

Massimo Benuzzi 

Presidente ASSOMETAB (Associazione Italiana dei produttori e distributori di mezzi tecnici per l’agricoltura biologica 

e ecocompatibile) 

L’Agricoltura biologica è uscita da una fase pionieristica nella quale le cosiddette “scelte di vita” avevano maggior 

importanza nella gestione dell’azienda rispetto a decisioni di tipo imprenditoriale; l’incremento delle superfici 

interessate e l’esigenza di soddisfare la sempre più crescente domanda di “cibi puliti” pone il settore di fronte a precise 

scelte. Anche la Grande Distribuzione Organizzata ha recentemente dimostrato che il prodotto biologico può entrare 

nella sua gamma, promuovendo anche marchi propri che contraddistinguono una specifica linea di agricoltura 

biologica. Nello stesso tempo diventa sempre più chiaro che i prodotti da agricoltura biologica potranno ulteriormente 

crescere in quantità e qualità, con le adeguate remunerazioni per tutti i soggetti della filiera, solo se verranno garantite in 

modo sicuro e trasparente la certificazione delle produzioni biologiche. 

Senza entrare nel merito del “controllo dei controllori” o dell’esistenza o meno di problemi nella catena di 

certificazione, ASSOMETAB vuole mettere in evidenza quanto al momento attuale sia stato poco considerato il tema 

dei mezzi tecnici per l’agricoltura biologica. 

Prima di tutto è necessario sottolineare che Istituti di ricerca e Università stanno, attraverso le aziende interessate, 

sempre di più proponendo nuove soluzioni ai problemi tecnici che scaturiscono dalle esigenze degli operatori del 

biologico. Infatti negli ultimi anni sono stati risolti numerosi problemi fitoiatrici, non solo legati all’agricoltura 

biologica, con l’avvento di nuovi insetticidi e fungicidi di origine naturale, soluzioni tecniche alternative alla chimica. 

Fondamentale è il ruolo delle aziende legate a questo settore che hanno investito e stanno investendo nella ricerca, nella 

messa a punto di nuovi biopesticidi (ma anche di fertilizzanti) e anche nelle relative registrazioni. Si riesce così a 

coniugare economia e ecologia, ovvero due cose che, fino a poco tempo fa, sembravano inconciliabili. Se non ci fossero 

aziende indirizzate a questo settore dell’agricoltura probabilmente i produttori biologici dovrebbero attendere che anche 

le multinazionali agrochimiche decidano di muoversi per promuovere lo sviluppo del settore. Forse, in quel caso, 

dovrebbero armarsi di tanta pazienza…… 

Il fatto che in Italia sia presente una Associazione come ASSOMETAB con aziende che investono nella ricerca di 

fertilizzanti e fitofarmaci per il biologico proprio nel paese europeo con le superficie più ampia a biologico, forse, non è 

un caso. 

Anche gli operatori del biologico (agricoltori e tecnici) devono avere presente che scegliere aziende che investono nel 

biologico (come quelle di ASSOMETAB) significa dare una mano a soggetti che sicuramente reinvestiranno buona 

parte dei ricavi in nuovi prodotti per il biologico, innescando così un circolo virtuoso; magari un concime o un Bacillus 

thuringiensis di una multinazionale sono più economici, ma in un’ottica più lungimirante (quella che fa crescere un 

settore), privilegiare una azienda rispetto ad un altra equivale ad effettuare una scelta che potrebbe rivelarsi importante 

per lo sviluppo dl settore. 

Questo sviluppo è però attualmente minacciato dal Decreto 290, più specificamente dai punti 1 e 2 dall’articolo 38 che 

liberalizzando in modo assolutamente inadeguato il settore ha permesso ad aziende con poche scrupoli di mettere sul 

mercato mezzi tecnici non testati che possono screditare tutto il segmento. 

Riportiamo alcuni esempi di quali problemi tale DPR ha scatenato: 

- Come potranno gli enti certificatori garantire che i prodotti biologici siano sicuri quando i fitofarmaci per 

produrli non sono adeguatamente controllati ? 

- Come potrà la GDO garantire che i propri marchi siano realmente aderenti a quanto vantato nelle promozioni 

pubblicitarie ? 

- Perché aziende che hanno investito ingenti somme in registrazioni si devono trovare fronteggiare una 

concorrenza “sleale” che mette sul mercato fitofarmaci prodotti e controllati da “apprendisti stregoni” ? 

- Perché quando un insetticida biologico non è registrato su una coltura, quello simile (o che per lo meno vanta 

di esserlo con il nuovo DPR) può essere invece impiegato dovunque ? In pratica per i fitofarmaci registrati 

esiste la necessità di registrare i prodotti su ciascuna coltura indicando anche i residui ammessi; nel caso di 

prodotti “regolarizzati” con il DPR 290 invece non si pone alcun limite su nessuna coltura, creando di fatto una 

disparità assolutamente assurda. 

- Perche il DPR 290 non specifica assolutamente niente circa i tempi di carenza; forse il rotenone regolarmente 

registrato deve avere 10 giorni di tempo di carenza e quello ammesso dal DPR 290 nessun giorno? I Bacillus 

thuringiensis registrati 3 e i Bacillus thuringiensis non passati al vaglio del Ministero della Salute nessuno ? 

Significa che è meglio non farsi controllare ? 

Questi sono alcuni punti che vorremmo far sapere a chi si interessa veramente del settore. 

ASSOMETAB ha avanzato al MIPAF sin dall’estate scorsa le sue proposte per una normativa chiara ed efficace sui 

mezzi tecnici per l’agricoltura biologica. In sintesi una registrazione semplificata che abbrevi significativamente i 

lunghi tempi (e i relativi costi) prima dell’immissione sul mercato, ma che mantenga elevate garanzie per il 

consumatore, l’agricoltore e l’ambiente. 


41

IMPEGNO DEL MIPAF NEL BIOLOGICO 

Giancarlo Imbroglini 

In questi ultimi tempi, si è osservata una notevole crescita dell’agricoltura biologica in Italia che ha portato il nostro 

Paese a detenere il primato in Europa per numero di aziende e per superficie coltivata a biologico ed a collocarsi al 3° 

posto nel mondo. Chi coltiva, alleva, prepara e importa, secondo il metodo di produzione biologico, deve sottostare alle 

disposizioni contenute nel Regolamento della Comunità Europea n. 2092/91 e successive modifiche e integrazioni. 

Nell’allegato II B del suddetto Regolamento sono elencati i prodotti fitosanitari che possono essere impiegati per la 

difesa delle colture. I prodotti ammessi sono, però, in numero esiguo, dotati, generalmente, di scarsa efficacia e inoltre, 

per alcuni di essi, quali i sali di rame, sono stati fissati limiti massimi di impiego. Questa situazione concorre fortemente 

a frenare la crescita delle produzioni biologiche. Gli approfondimenti scientifici che si rendono, pertanto, necessari 

devono tendere a fornire risposte esaustive alle numerose problematiche che gli operatori del biologico si trovano a 

dover affrontare ed a vagliare con cura le innovazioni strategiche per la difesa non chimica delle colture. La ricerca in 

agricoltura biologica è caratterizzata da una maggiore complessità rispetto all’agricoltura convenzionale in quanto la 

gestione della produzione si fonda su sistemi olistici che rendono necessaria l’armonizzazione delle pratiche 

agronomiche nel loro complesso. Anche la difesa fitosanitaria va inquadrata in un’ottica di sistema complesso e non 

come verifica del miglior prodotto per risolvere un problema specifico. Nell’ambito delle attività di tutela e sviluppo 

dell’agricoltura biologica, il Ministero delle Politiche Agricole e Forestali (MiPAF), autorità di controllo del settore a 

livello nazionale, ha promosso e sostiene un programma per la trattazione delle diverse tematiche che riguardano la 

difesa delle piante coltivate secondo il metodo biologico con riferimento sia agli aspetti normativi che agli aspetti 

sperimentali. Ha cercato, in tal modo, di supportare l’agricoltura biologica con finanziamenti specifici. Con il progetto 

“Difesa delle Produzioni in agricoltura biologica”, di durata triennale, ci si è posti l’obiettivo di investigare sui mezzi 

tecnici utilizzabili per la difesa in agricoltura biologica valutandone l’efficacia, cercando di mettere a punto i dosaggi 

ottimali, le epoche di trattamento più convenienti, il numero di trattamenti, nonché gli eventuali effetti secondari a loro 

ascrivibili. L’obiettivo è anche quello di individuare altri mezzi di difesa, compatibili con il metodo di produzione 

biologico, in grado di contrastare le numerose avversità di pre e post-raccolta. 


42

POSTER 

1. Agosteo G.E., Ambrosio M.: Prove di lotta all'oidio dello zucchino con prodotti alternativi in 

pieno campo 

2. Arnoldi A., D'Agostina A., Boschin G.: Lupinus Albus, an ancient European legume 

particulary suitable for organic farming, may become an useful source of functional ingredients 

3. Barrese E., Benincasa C., Lombardo N., Mazzalupo I., Pellegrino M., Perri E., Sindona 

G.: Benzo(a)pyrene in organic virgin olive oils 

4. Cabizza M., Melis M., Cabras P.: Effetto delle cere epicuticulari di frutta e vegetali sulla 

fotodegradazione del rotenone 

5. Castagnoli M., Liguori M., Nannelli R., Simoni S.: Insetticidi d'origine vegetale e acari 

6. Casucci C., Monaci E., Perucci P.: Changes of biochemical parameters in soil amended with 

moist olive husks 

7. Casulli F., Santamauro A., Tauro G., Gatto M.A., Faretra F.: Natural compounds in the 

control of powdery mildew on cucurbits in organic agriculture 

8. Citarrei F., Scribano M., Coranelli S., Cellerino C., Quattrocchi L., Concezzi L., Ragni A.: 

Methods for evaluation, in controlled conditions, of the growth of oilseed rape promoted by 

bacteria 

9. Convertini G., Donato F., Sasanelli N., D'Addabbo T.: Effetto dello spandimento di residui 

dell'industria olearia sulla fertilità del suolo e su nematodi fitoparassiti 

10. D'Addabbo T., Sasanelli N.: Nematicidal activity of acqueous extracts from rue (Ruta 

Graveolens L.) 

11. Gargani E., Del Bene G.: Valutazione dell'efficacia insetticida di Ryania, Rotenone, 

Azadiractina e Beauveria Bassiana 

12. Dugo G.mo, Giuffrida D., Drogo A., La Pera L.: Determination of Cd (II), Cu (II), Pb (II) 

and Zn (II) in biological and not biological citrus essential oils by derivative potentiometric 

stripping analysis (dPSA) 

13. Floris I., Satta A., Cabras P., Angioni A.: Impiego del timolo nel controllo della varroosi delle 

api. Efficacia, persistenza e residui 

14. Pertot I., Delaiti M., Forti D.: Assesment of phytotoxicity to Grapevine of traditional and new 

copper compounds used in copper reduction strategies in organic viticulture and its relationship 

with environmental conditions and number of treatments 


43

15. Fusari F., Petrini A.: Organic wheat quality and production: the results of three years of trials 

16. Iannotta N.: Il controllo della "mosca delle olive" (Bactrocera oleae Gmel.) con metodi 

consentiti in coltivazione biologica 

17. Kelderer M., Casera C., Lardschneider E.: Il contenimento della ticchiolatura in frutticoltura 

biologica 

18. La Pera L., Lo Turco V., Lo Curto S., Mavrogeni E., Dugo G.mo: Influence of different 

treatments on Cd (II), Cu (II), Pb (II) and Zn (II) content in Sicilian olive oils 

19. La Torre G.L., Pellicanò T.M., Pollicino D., Alfa M., Dugo G.mo: Determination of phenolic 

compounds in experimental wines subjected to different pesticides treatments 

20. La Torre A., Donnarumma L., Lolletti D., Imbroglini G.: Control of apple scab in organic 

farming 

21. Maietti A., Mazzotta L., Saletti C., Mirolo G., Berveglieri M., Tedeschi P., Brandolini V.: 

Contaminazione da micotossine in alimenti di produzione biologica 

22. Maini P.: Peptidati di rame: prodotti innovativi a basso dosaggio a base di rame, chelato ad 

amino acidi e peptidi 

23. Perri E., Lombardo N., Muzzalupo I., Urso E., Pellegrino M., Sindona G., Benincasa C., 

Cavallo C.: Characterization of organic virgin olive oils from coratina cultivar 

24. Perri E., Mazzalupo I., Rizzuti B., Pellegrino M., Sindona G., Benincasa C., Cavallo C.: 

Characterization of organic virgin olive oils from ogliarola salentina cultivar 

25. Pertot I., De Luca F., Vecchione A., Zulini L.: Efficacy evaluation of biological control 

agents against Plasmopara viticola 

26. Pulga A., Valmori I.: Il controllo delle poerazioni di campo come base della rintracciabilità a 

garanzia del consumatore e dell'ambiente 

27. Ricci M., Colli M., Barcarotti R., Ragni A.: Eco-toxicity of the entomopathogenic bacteriumnematode 

symbiotic complex toward non-target organisms 

28. Ragni A., Valentini F.: Photorhabdus e Xenorhabdus: a new source of useful compounds for 

biological control 

29. Ricci M., Flek G., Colli M., Barcarotti R., Quattrocchi L., Coranelli S., Concezzi L., Fifi 

A.P., De Nicola R., Scribano M., De Berardinis M., Citarrei F., Ragni A.: Bioassays for 

screening and quality control of products with insecticidal, fungicidal and nematocidal activity, 

based on plant extracts, microorganisms and chemical molecules 

30. Scribano M., Ragni A.: Isolation of new biological control agents from Umbria region 

terrotory 


44

31. Pietri A., Bertuzzi T., Barbieri G., Rossi F.: Protein content and mycotoxins contamination in 

organic and conventional wheat 


45

32. Tedeschi P., Maietti A., Mazzotta D., Vecchiati G., Romano P., Brandolini V.: 

Determinazione di residui metallici nelle pere e influenza sull'impatto ambientale 

33. Varvaro L., Antonelli M., Balestra G.M., Fabi A., Scermino D., Vuono G.: Investigations 

on the bactericidal activity of some natural products 

34. Cravedi P., Molinari F. :Ricerca su insetticidi utilizzabili secondo il metodo di produzione 

biologico di prodotti agricoli 

35. Salvo F., Saitta M., Di Bella G., La Pera L., Dugo G.mo: Fungicides and heavy metals in 

wine samples produced from experimental grapes subjected to different pesticides treatments 

36. Capasso R., De Martino A., Cristinzio G., Di Maro A., Parente A.: New α-elicitin isoforms 

from Phytophthora hybernalis as protein elicitors for a potential employment in the biological 

pest mangement 


46

1 

PROVE DI LOTTA ALL’OIDIO DELLO ZUCCHINO CON PRODOTTI 

ALTERNATIVI IN PIENO CAMPO 

G.E. Agosteo, M. Ambrosio 

Dipartimento di Agrochimica ed Agrobiologia - Università Mediterranea di Reggio Calabria 

Premessa 

La coltura dello zucchino in Calabria è ampiamente diffusa sia pieno campo che in ambiente protetto. L’intensità delle 

infezioni di oidio (Sphaerotheca fusca Blumer, Erysiphe cichoracearum D.C.) su questa coltura è particolarmente 

elevata. A differenza del melone che dispone di un’ampia gamma di varietà resistenti o tolleranti alla malattia, per lo 

zucchino è necessario ricorrere a ripetuti interventi di lotta chimica, pena il precoce decadimento vegetativo delle 

piante. La gestione della lotta all’oidio, per il rispetto degli intervalli di sicurezza dei fungicidi, è complicata dalla 

scalarità della raccolta. Lo zolfo, unico fungicida antioidico autorizzato in agricoltura biologica, può dare problemi di 

selettività su alcune varietà di cucurbitacee, soprattutto in presenza di temperature elevate, quali quelle del periodo 

primaverile-estivo nel meridione d’Italia. 

Con la presente prova si è inteso saggiare l’efficacia antioidica su zucchino in pieno campo, in un ambiente di 

produzione meridionale caratterizzato da forte pressione di malattia, di alcuni prodotti di origine minerale, quali 

bicarbonato di sodio e fosfato monopotassico, il cui impiego è stato sperimentato con successo in altri ambienti di 

coltivazione (Minuto et al., 1998). 

Materiali e metodi 

La prova è stata realizzata nell’anno 2001, nella Piana di Lamezia Terme (CZ), su zucchino, cv. Clarita, allevato in 

pieno campo, con un ciclo colturale di circa 70 giorni. Il trapianto è stato realizzato il 25 maggio, le piante hanno 

occupato il terreno in un periodo stagionale (giugno – luglio) caratterizzato da temperature particolarmente favorevoli al 

patogeno. E’ stato utilizzato uno schema sperimentale a blocchi randomizzati con 7 tesi (Tab.1) e 4 ripetizioni. 

Tab.1 - Tesi a confronto, dosi, intervalli tra le applicazioni 

Tesi Principio attivo 

Nome commerciale e/o p.a. (%) e Dosi (ml o g/hl) Intervallo tra 

società produttrice formulazione p.c. p.a. le applicazioni 

1 Zolfo bagnabile Zolfo bagnabile - Isagro 90 Pb 500 450 7 giorni 

2 Bicarbonato di sodio Solvay 100 Pb 750 750 7 giorni 

3 Bicarbonato di sodio Solvay 100 Pb 1000 1000 7 giorni 

4 KH2PO4 (0-52-34) Haifa Chemicals Ltd 100 Cris.sol. 1000 1000 7 giorni 

5 Trifloxystrobin Flint - Bayer 50 WG 25 12,5 14 giorni 

6 

Zolfo bagnabile, 

penconazole 

Zolfo bagnabile - Isagro 

Topas 10 EC - Syngenta 

90 Pb 

10,52 Le 

500 

35 

450 

3,5 

7 giorni 

14 giorni 

7 Testimone \ \ \ \ \ 

Ciascuna parcella era costituita da 12 piante contigue sulla fila. I trattamenti hanno avuto inizio l’11 giugno, prima della 

comparsa dei sintomi, e sono stati effettuati con le cadenze riportate nella tab.1. Nella tesi 6 (trattamento aziendale), 

sono stati realizzati due interventi con zolfo seguiti, alla comparsa dei sintomi, dal penconazole. La lettura dei sintomi è 

stata realizzata a 42 e 60 giorni dall’inizio del ciclo, su 4 foglie per pianta (1 dei palchi basali, 2 dei palchi medi ed 1 

apicale), da 5 piante centrali a parcella, sulla base di una scala di malattia con 6 classi d’infezione (0=0; 1=0-1, 2=1-5, 

3=5-20, 4=20-40, 5= >40 % di area fogliare infetta) (EPPO, 1997). 

Sono stati determinati gli indici di diffusione (percentuale di foglie infette), gravità G = Σ (f . v) n -1 ed intensità (indice 

di McKinney) (I = [Σf . v (N . X) -1 ] . 100), dove f = frequenza dei casi per ciascuna classe d’infezione, v= valore di classe 

corrispondente, n = numero di casi infetti, N = numero totale delle osservazioni, X = valore massimo della classe nella 

scala d’infezione. I dati sono stati sottoposti ad analisi della varianza, le medie sono state separate con il test di Duncan 

per P=0,05 e P=0,01. I valori percentuali sono stati trasformati nei corrispondenti valori angolari. 


47

Risultati 

I risultati conseguiti sono riportati nella tab.2. 

Tab.2 – Indici di malattia a 42 e 60 giorni dall’inizio del ciclo colturale 

Tesi 

Rilievo a 42 gg 

Diffusione Gravità Intensità 

Rilievo a 60 gg 

Diffusione Gravità Intensità 

1. Zolfo bagnabile 86,2 b AB 3,52ab A 60,7 b B 98,7 a AB 4,61ab AB 91,5 b AB 

2. Bicarb. di sodio 750 g/hl 83,7 b BC 3,45 ab A 57,2 bc BC 98,7 a AB 4,53 bc BC 89,5 bc B 

3. Bicarb. di sodio 1000 g/hl 78,7 bc BC 3,52 ab A 57,6 bc BC 96,2 ab AB 4,47 bcde BC 86,25 c B 

4. KH2PO4 1000 g/hl 83,7 b BC 3,20 b A 52,7 cd BC 100 a A 4,51 bcd BC 89,75 bc B 

5. Trifloxystrobin 26,2 d D 1,35 c B 7,5 eD 92,5 bc BC 4,32 de C 80 d C 

6 .Zolfo, Penconazole 75,0 c C 3,40 ab A 50,7 d C 97,5 ab AB 4,39 cde BC 86,75 c B 

7. Testimone 96,2 a A 3,72 a A 69,7 a A 100 a A 4,77 a A 95,5 a A 

Le medie sono state separate secondo il test di Duncan. Valori seguiti da stesse lettere non differiscono 

significativamente fra loro per P= 0,05 (lettere minuscole) e P= 0,01 (lettere maiuscole) 

La pressione di malattia è stata molto elevata (96% e 100% di foglie infette nel testimone, rispettivamente, nei due 

rilievi). Alla data del 1° rilievo il trifloxystrobin, strobilurina di sintesi, ha fatto rilevare indici di malattia molto 

contenuti, con infezioni limitate alle sole foglie basali. Tutte le altre tesi si sono differenziate statisticamente dal 

testimone ma con indici di malattia elevati. Il fosfato monopotassico ha fatto rilevare una migliore attività antioidica 

rispetto allo zolfo ed un indice complessivo di malattia (intensità) non differente statisticamente dalla tesi di riferimento 

aziendale (zolfo, penconazole). Il bicarbonato di sodio (sia allo 0,75 che all’1%) ha mostrato un’attività analoga a 

quella dello zolfo. 

In prossimità della fine del ciclo colturale (2° rilievo) le differenze fra le tesi si sono notevolmente ridotte ed il 

contenimento della malattia è stato insoddisfacente. L’intensità di malattia è stata statisticamente differente dal 

testimone in tutte le tesi, grazie soprattutto ad un minore peso della gravità. Il trifloxystrobin ha mostrato i valori di 

intensità più contenuti e, unico fra le tesi saggiate, una diffusione statisticamente differente dal testimone. Il bicarbonato 

di sodio all’1% ha fatto rilevare un’intensità minore rispetto allo zolfo, in linea con la tesi aziendale (zolfo, 

penconazole). Alle dosi utilizzate non sono stati osservati effetti fitotossici sulle piante. 

Conclusioni 

Un soddisfacente contenimento delle infezioni di oidio su zucchino, negli ambienti di coltivazione meridionali 

caratterizzati da forte pressione di malattia, richiede l’impostazione di programmi di lotta che facciano ricorso a 

fungicidi con azione curativa. Fosfato monopotassico e bicarbonato di sodio hanno evidenziato un’attività significativa, 

migliore od analoga a quella dello zolfo, di cui costituiscono pertanto una valida alternativa in applicazioni di tipo 

preventivo. 

Lavori citati 

EPPO, 1997. Powdery mildew of cucurbits and other vegetables, PP 1/57(3). EPPO Standards. Guidelines for the 

efficacy evaluation of plant protection products. Vol. 2, EPPO Parigi, 81-85. 

MINUTO A., MINUTO G., GULLINO M.L., GARIBALDI A., 1998. Prove di lotta al mal bianco dello zucchino. In: 

Atti Giornate Fitopatologiche, 655-660. 


48

2 

LUPINUS ALBUS, AN ANCIENT EUROPEAN LEGUME PARTICULARLY SUITABLE FOR ORGANIC 

FARMING, MAY BECOME AN USEFUL SOURCE OF FUNCTIONAL INGREDIENTS 

Anna Arnoldi, Alessandra D’Agostina, Giovanna Boschin 

DISMA, Section of Chemistry – University of Milan (anna.arnoldi@unimi.it) 

Lupin is a protein rich grain legume typical of the Mediterranean region that produces seeds with a protein content, both 

qualitatively and quantitatively similar to that of soybean. A very interesting advantage of lupin in respect to soybean is 

that there are no commercially available genetically modified varieties, which makes it an ideal crop for organic 

farming. In addition it is particularly valuable in Mediterranean areas, owing to its drought resistant properties, and, 

being a legume, it may be used in rotation with other cultures to reduce the use of fertilisers. For these reasons the 

European Commission has included it in the list of plants that merit specific research efforts (1). 

The protein content of lupin, varying from species to species, is around 35 to 40% in Lupinus albus. About 10% of the 

total protein content consists of albumins, while the rest is represented by globulins, showing a 1:1 legumin 

(11S)/vicilin (7S) ratio. Ultracentrifugation of the protein solutions leads to 4 fractions with sedimentation coefficients 

of 15S, 11S, 7S and 2S. The major fractions are the 11S and the 7S fractions, which account for about 33 to 37% of 

total proteins (2). In addition, lupin contains a specific protein fraction, conglutin γ, which accounts for about 5% of 

total proteins and has the exceptional characteristic of being a sulphur-rich protein, thus containing amino acids that are 

scarce in other grain legumes. 

In respect to soybean, lupin kernels contain lower amounts of antinutrients, potentially of consumer concern, when 

using protein ingredients in food preparations. Trypsin inhibitor concentration is, in fact, 0.18 mg/g in lupin vs. 17.90 

mg/g in soybean, phytate concentration is 0.44 mg/g vs. 1.59 mg/g, saponin concentration is 1.4 mg/g vs. 1.9 mg/g, and 

the concentration of oligosaccharides of the raffinose family is 4.6 mg/g vs. 5.7 mg/g (3). Lupin alkaloids, which, also 

for their bitter taste, represented a strong limitation for the human consumption of some old lupin varieties, have now 

been reduced to very low levels in the “sweet lupin” varieties (4). Sweet lupin flour has been suggested for use in bread, 

cookies and milk substitutes. Manufacturers advertise that these products have an increased dietary fibre content and 

high protein value. Additionally lupin seeds do not contain phyto-hormones, in particular isoflavones that, according to 

recent literature data, have serious toxicological potentials. 

In the last decade, the increasing awareness of the health consequences of an incorrect diet and some scandals in the 

meat market (BSE, epizootic aphtha, contamination with polychloro-dibenzodioxines and/or furans residues) have 

encouraged the consumers to go back to legumes as a source of valuable proteins. In this situation, lupin seeds have all 

the characteristics to become one of the main source of vegetable food ingredients in the Mediterranean area. 

1. European Economic and Social Committee, Section for Agriculture, Rural Development and the Environment, 

‘New impetus for a plan for plant protein crops in the Community’, Brussels, 4 December 2001. 

2. Guegen J, Cerletti P. In New and Developing Sources of Food Proteins. Hudson, BJF ed., Chapman and Hall, 

London 1994, 145-193. 

3. Champ M. Proceedings 4th Europ. Conference on Grain Legumes, 2001, 109-113. 

4. Muzquiz M, Pedrosa MM, Cuadrado C, Ayet G, Burbano C, Brenes A In Recent Advances of Research in 

Antinutritional Factors in Legume Seeds and Rape Seeds. Jasman AJM et al eds, EAAP Publication N. 93, 

Wageningen Press, 1998, 387-390. 


49

3 

BENZO(a)PYRENE IN ORGANIC VIRGIN OLIVE OILS 

a Elena Barrese, b Cinzia Benincasa, a Nicola Lombardo, b Fabio Mazzotti, a Innocenzo Muzzalupo, a Massimiliano 

Pellegrino, a Enzo Perri, b Giovanni Sindona 

a) Istituto Sperimentale per l'Olivicoltura, 87030 Rende (CS)-Italy 

b) Dipartimento di Chimica, Università della Calabria, via P. Bucci, cubo 15/c I -87030 Arcavacata di Rende (CS)-Italy 

Introduction 

Polycyclic aromatic hydrocarbons (PAHs) are environmentally hazardous organic compounds because of their known 

or suspected carcinogenicity. PAHs are mostly formed during an incomplete combustion of organic material and they 

are known as highly stable contaminants present in many foods. This contamination can be a result of sorption from the 

environment or from food preparation methods. Air pollution is the main source of PAHs in the edible parts of plants. 

Hence, the determination of PAHs in foods such as olives and olive oil is important for human health. Benzo(a)pyrene 

(BaP) and Benzo(e)pyrene (BeP) are the best known PAHs and they have been often used as indicators of the presence 

of PAHs in foods. Several authors have reported the presence of Benzopyrenes (BP) in edible oils and fats. However, 

there are few reports on the BP content in olive oils (Mariani et al., 1984; Menichini et al., 1991; Fiume et al.2002) 

while there has been no reports on the BP content in organic virgin olive oils. In general, the detected levels of BP in the 

olive oils analyzed were in the range of a few µg/kg of olive oil (Pupin et al., 1996; Vazquez Troche et al., 2000). 

However, olive oil refining involving deodorization, bleaching and charchoal treatment reduces the content of PAHs. 

Therefore, paradoxically, higher levels of BP may be found in virgin olive oils, irrespective of farming system adopted, 

since they are, by definition, unrefined and untreated. 

Currently no legislation exists regarding BP levels in vegetable oils in European Union. However, the International 

Olive Council (IOC), the German food industries and some European nations (Italy, Spain and Portugal) recommend 

for olive-pomace oil and refined olive-pomace oil a BaP maximum level of 2 µg/kg. Therefore, there is a need to verify 

the level of BP in European olive oils before a regulation occur. The goal of this preliminary work was to assess the 

presence and determine the level of BP in organic virgin olive oils from the most important olive growing region of 

Italy: Apulia. 


Plant material. In 2000/2001 harvest, samples of organically cultivated olive drupes of Coratina cv from a farm at 

Andria (Bari, Italy) were collected 10 days before the traditional start of harvest, in the middle of the harvest period and 

10 days after the end of harvest time. The olives were handpicked each time from 5 olive tree of the same cultivar. 

Work-up of plant material. Olive drupes (10 Kg) were crushed with a hammer mill and the oil was extracted by 

centrifugation after 20 minutes of malaxation at RT. 

Analytical procedures.Extraction and clean-up procedure were conducted by slightly modifying Vazquez Troche et al. 

(2000) protocol. The final extract was analyzed by a Hewlett Packard HP 5973 Mass Spectrometer interfaced with a HP 

6890A GC (Agilent Technologies, Waldronn, GmbH) and BaP detected as molecular ion (m/z 252) by Selected Ion 

Monitoring (SIM) mode. Quantification was obtained by the external standard method. 


Detection limit for BP in the acetonitrile eluate was 0.3 µg/kg of olive oil. The standard calibration curves obtained with 

a linear coefficient of 0,999 generated from triplicate 1 µl injections at different concentration show a linear range from 

0.3 µg/kg to 5.4 µg/kg with good-to-excellent R 2 values. For reproducibility test three concentrations were analyzed and 

the average coefficient of variation (CV%) was 5%. The recovery obtained for spiked samples with 4 µg/Kg of BaP was 

66%. As expected, also the organic olive oils are affected with a PAHs. In fact, the levels of BaP and BeP in organic 

virgin olive oils from Coratina cv. at Andria (Apulia) ranged from 0.39 and 0.85 µg/kg, and 0.38 and 0.60 µg/kg , 

respectively, while the average content was 0.56 µg/Kg and 0.52 µg/Kg, respectively (table 1). Although the BP values 

were low, the determination of the other “heavy” PAHs is still required to assess the real intakes of PAHs from the total 

diet of apulian olive oil consumer. Therefore, the determination of the most important “heavy” PAHs in organic virgin 

olive oil by GC-MS is in progress. 


50

Table 1. Benzo(a)pyrene and benzo(e)pyrene content in organic virgin olive oils from Coratina cv. at Andria 

(Apulia). 

Cultivar Origin Harvest time BaP (µg/Kg of olive oil) BeP (µg/Kg of olive oil) 

Coratina Andria (Apulia) 11/07/00 0.85 0,38 



Mean + Std. dev. 0.56+ 0.20 0.52+ 0.10 

Acknowledgements 

We thank Dr. Cosimo Cavallo (Regione Puglia), Dr. Nino Paparella (C.I.Bi., Bari), Dr. Nicola Panaro (C.I.Bi., 

Bari) and Drs. Edoardo and Giancarlo Ceci Ginistrelli for olive sampling, data collection on farming systems and olive 

grove management. We thank also Prof. Angelo Putignano and Prof. Francesco Prudentino (I.T.A.S. of Ostuni-BR) for 

olive oil extraction. 

References 

F. Fiume, F. Ferrieri, G. Froio, S. Spinello, O Lattarulo, G. Fanuzzi, 2002, Determinazione di idrocarburi policiclici 

aromatici in oli alimentari, Riv. Ital. Sostanze grasse, LXXIV, 151-155. 

C. Mariani, E. Fedeli,1984, Idrocarburi policiclici aromatici negli oli vegetali, Riv. Ital. Sostanze Grasse, 61, 305 -315. 

E. Menichini, A. Bocca, F. Merli F., Ianni, F. Monfredini, 1991, Polycyclic aromatic hydocarbons in olive oils on the 

italian market, Food Addit. Contam., 8(3), 363-369. 

A. M. Pupin and Maria Cecilia Figueiredo Toledo, Benzo(a)pyrene in olive oils on the brazilian market, Food 

Chemistry, 1996, 55(2), 185-188. 

S. Vazquez Troche, M.S. Garcìa Falcòn, S. Gonzales Amigo, M.A. Lage Yusty, J. Simal Lozano, Enrichment of 

benzo(a)pyrene in vegetable oils and determination by HPLC-FL, Talanta 2000, 51, 1069-1076. 


51

4 

EFFETTO DELLE CERE EPICUTICULARI DI FRUTTA E VEGETALI SULLA FOTODEGRADAZIONE 

DEL ROTENONE 

M. Cabizza, M. Melis, P.Cabras 

Dip. di Tossicologia Università di Cagliari 

Via Ospedale, 72 Cagliari 

E' stato studiato l'effetto delle cere epicuticulari di frutta e ortaggi sulla fotodegradazione del rotenone alla luce solare 

La fotodegradazione è stata condotta utilizzando capsule di petri all'interno delle quali veniva posto il rotenone con (o 

senza) la cera di susine, nettarine, mele, pere, melanzane e pomodoro. Le capsule sono state esposte alla luce solare e i 

campioni prelevati a intervalli di tempo prestabiliti Dopo l'esposizione al sole i campioni sono stati ripresi con 

acetonitrile:acqua 50:50 (v/v), iniettati per l'analisi HPLC e sono stati determinati il rotenone e i suoi principali 

metaboliti: 6',7'-epoxy-rotenone α e β, rotenolone 6αβ,12αα, rotenolone 6αβ,12αβ, deidrorotenone. 

Il tempo di semivita del rotenone irradiato alla luce solare senza cere era 1,0 ore. In presenza di cere di pomodoro non 

c'erano sensibili variazioni mentre con cera di nettarine e susine il tempo di semivita risultava maggiore. Le cere di 

mele, pere e melanzane aumentavano la cinetica di fotodegradazione del p.a. 

Il rotenone irradiato al sole in assenza di cere formava 5 metaboliti più importanti (6',7'epoxy-rotenone α e β, 

rotenolone 6αβ,12αα, rotenolone 6αβ,12αβ, deidrorotenone e un metabolita con tr 5,37) mentre irradiando il rotenone 

in presenza di cere si avevano differenze nella formazione di tali metaboliti sia dal punto di vista qualitativo che 

quantitativo. Solo irradiando il rotenone in presenza di cere di pomodoro tali metaboliti si formavano 

contemporaneamente mentre con tutte le altre cere impiegate esistono differenze nella loro formazione a seconda del 

tipo di cera utilizzato. La cera di pere inibisce la formazione di tutti i metaboliti ad eccezione del deidrorotenone e del 

rotenolone 6αβ,12αβ. 

Il metabolita rotenolone 6αβ,12αβ si forma sempre nel rotenone irradiato sia in assenza che in presenza di cere ed è il 

metabolita quantitativamente più abbondante. 


52

5 

INSETTICIDI D’ORIGINE VEGETALE E ACARI 

Castagnoli M., Liguori M., Nannelli R., Simoni S. 

Sezione di Acarologia, Istituto Sperimentale per la Zoologia Agraria. Firenze 

Different formulations of extracts of neem, derris, equisetum, ryania, wormwood, garlic, chrysanthemum, and 

bitter wood were tested on the phytophagous mite Tetranychus urticae, the phytoseiid Neoseiulus californicus, the 

stored food mite Lepidoglyphus destructor to evaluate their different toxicity on eggs and females and the effects on the 

female fecundity. 

On the whole, these botanical pesticides were more effective on eggs than on motile stages. The highest 

toxicity was registered on N. californicus. 

The extracts based on low concentrations of azadirachtina and neem oil were the most toxic for the females and 

the eggs of the tetranychid and did not significantly affect the survival and fecundity of the phytoseiid. Derris and 

wormwood extracts were the most effective on the stored food mite. 

KEY WORDS: Tetranychus urticae, Neoseiulus californicus, Lepidoglyphus destructor, neem, derris, ryania, wormwood, 

chrysanthemum, bitter wood. 

INTRODUZIONE 

Negli ultimi anni una maggiore sensibilità verso le conseguenze negative per l’ambiente determinate dall’uso 

di molti pesticidi di sintesi ha determinato anche nel nostro Paese una crescita di interesse nei confronti dei prodotti 

d’origine vegetale ritenuti più facilmente degradabili. L'uso di questi prodotti commercializzati sia come insetticidi o 

più genericamente come biostimolanti e coadiuvanti ad azione limitatrice nei confronti di artropodi nocivi si è 

particolarmente affermato in agricoltura biologica. Nell'ottica di una valutazione complessiva dei loro effetti, si rende 

quindi sempre più necessaria la verifica anche sulle specie non target. 

L’efficacia di sostanze naturali come rotenone, piretro e quassia è nota da tempo, più recente è l’interesse per i 

derivati di Azadiracta indica (A. Juss) ritenuti in grado di esplicare azione tossica, repellente, fagodeterrente e inibitrice 

della crescita su moltissime specie di artropodi (SCHMUTTERER, 1990). Per quanto riguarda gli acari le conoscenze sono 

molto limitate e, se poche sono le ricerche dedicate all'attività biologica di estratti di neem (MANSOUR et al.,1987,1997; 

SUNDARAN & SLOANE, 1995; SPOLLEN & ISMAN, 1996; MOMEN et al., 1997; PAPAIOANNOU-SOULIOTIS et al., 1997; 

TSOLAKIS et al., 1997), ancora meno sono quelle che prendono in esame altri principi di origine naturale (PERRUCCI, 

1995; GULATI & MATHUR, 1995; CASTAGNOLI et al., 2002). 

Nell' ambito di un più vasto programma di indagini, l’efficacia di alcuni principi attivi di origine vegetale è 

stata saggiata su tre delle più comuni specie di acari: Tetranychus urticae Koch, Neoseiulus californicus (McGregor) e 

Lepidoglyphus destructor (Schrank) (CASTAGNOLI et al., 2000; NANNELLI & SIMONI, 2001). Il primo è un fitofago 

estremamente polifago e in grado di causare seri danni su un’ampia gamma di colture, il secondo è un fitoseide 

predatore frequentemente associato al tetranichide e in grado di contrastarne le pullulazioni, mentre il terzo è un acaro 

astigmata che colonizza le più disparate derrate alimentari immagazzinate che vanno dai cereali ai salumi e ai formaggi. 

MATERIALI E METODI 

I prodotti saggiati e le relative diluizioni, che corrispondono alle dosi massime consigliate dai produttori, sono 

elencati nelle tabelle 1-3. In laboratorio sono stati valutati gli effetti sulla schiusura delle uova e sulla sopravvivenza 

delle femmine su un totale di 80 esemplari o uova per prodotto. Per il fitofago e il predatore è stata presa in 

considerazione anche l’incidenza sulla fecondità. 

I metodi usati erano diversificati a seconda dello stadio e delle caratteristiche della specie: “dip method” (HELLE & 

OVERMEER, 1985) e “microimmersion bioassay” (modificato da DENNEHY et al., 1993) sono stati usati rispettivamente 

per tutte le uova e per le femmine del fitofago e del predatore, mentre per le femmine dell’astigmata è stato usato il 

metodo "impregnated filter paper bioassay" secondo THIND & MUGGLETON (1998). 

I risultati sono stati espressi come percentuale di tossicità globale (E), combinando mortalità e fecondità delle 

femmine del trattato e del non trattato (OVERMEER & VAN ZOON, 1982) o come mortalità di ABBOTT (1925), tenendo 

conto dell’eventuale mortalità naturale registrata nel testimone. 


53

RISULTATI 

Derivati del neem (Azadirachta indica) 

In generale i prodotti saggiati sono risultati più efficaci sulle uova che sulle forme mobili delle tre specie. Per 

quanto concerne il fitofago, la maggiore efficacia sulle uova si è avuta con il prodotto in cui l'azadiractina era 

addizionata all'olio di neem e con quello in cui questo principio attivo era miscelato ad oli vegetali e lecitina di soia 

(tab.1, prodotti 1 e 2). Questi stessi formulati non determinano un incremento della fecondità di T. urticae come invece 

è stato osservato per gli altri prodotti. Sul predatore N. californicus l'azadiractina addizionata agli oli vegetali e lecitina 

di soia (tab.1, prodotto 2) oltre ad incidere pesantemente sulla schiusura delle uova determina la più alta mortalità delle 

femmine. Su L. destructor non si osserva una rilevante attività tossica di questo gruppo di prodotti; l'unico che 

determina un'apprezzabile riduzione della schiusura delle uova è quello con la più bassa percentuale di azadiractina 

(tab.1, prodotto 5). 

Piretrine 

Mostrano azione tossica sulle uova del fitofago e soprattutto su quelle dell'astigmata, ma non sulle femmine di 

queste due specie. Per quanto concerne il fitoseide l'azione varia dall'effetto positivo della formulazione con piretro al 

20% (tab.2, prodotto 1) a quello negativo del formulato con piretro al 25% (tab.2, prodotto 3). Ciò è imputabile quasi 

certamente alla diversa formulazione: in quest'ultimo le piretrine naturali sono miscelate ad oli vegetali, mentre il primo 

è una soluzione idroalcolica. 

Tabella 1. Estratti da neem (A = Azadiractina A): composizione e dose dei prodotti usati e loro effetto (E = tossicità 

globale, Abb. = mortalità secondo Abbott) su femmine e uova di due specie di acari di interesse agrario ( T. urticae e N. 

californicus) e di una specie di acaro delle derrate (L. destructor). 

Prodotti T. urticae N. californicus L. destructor 

composizione dose 

1 0,4% A + 0,09% 

olio di neem 

2 n. 1 + oli veget. 

e lecit. di soia 

3 1% A + olio di 

sesamo 

4 n. 3 + oli veg. e 

lecit. soia 

femmine 

E % 

uova 

Abb. % 

54 

femmine 

E % 

uova 

Abb. % 

femmine 

Abb. % 

uova 

Abb. % 

4g/l 12,98 100 15,00 9,08 -2,86 -4,35 

4g+2g/l 0,00 70,68 90,12 72,73 -3,90 17,39 

2,5g/l -50,86 20,05 4,88 33,76 -4,76 -26,09 

2,5g+2g/l -86,01 20,05 66,40 -2,59 -2,66 4,35 

5 0,15% A 3cc/l -17,56 20,05 -4,98 63,33 6,49 30,43 

6 3% A (Oikos) 1,5cc/l -16,65 15,94 8,94 0,00 12,50 21,74 

7 10% A 

(Neemazal) 

3g/l -17,87 28,99 35,50 -1,27 ---- ---- 


Tabella 2. Piretrine: composizione e dose dei prodotti usati e loro effetto (E = tossicità globale, Abb. = mortalità 

secondo Abbott) su femmine e uova di due specie di acari di interesse agrario (T. urticae e N. californicus) e di una 

specie di acaro delle derrate (L. destructor). 



femmine 

E % 

uova 

Abb. % 

55 

femmine 

E % 

uova 

Abb. % 

femmine 

Abb. % 

uova 

Abb. % 

1 piretro 20% 4,5cc/l -65,55 48,03 -23,37 -3,89 -8,42 91,30 

2 n. 1 + acidi grassi 

30% 

3 piretro 25% 

(Biopiren Plus) 

4,5cc+4g/l -18,04 10,72 -- -- 4,00 86,96 

1,6g/l -25,54 2,53 91,98 -1,27 ---- ---- 

Derivati da altre sostanze vegetali 

I derivati del derris manifestano tossicità soprattutto nei confronti delle femmine e delle uova del predatore e 

dell’astigmata, sul fitoseide la mortalità delle femmine supera con due prodotti il 90%. Sul fitofago il rotenone 5% (tab. 

3, prodotto 1) ha efficacia nei confronti delle femmine e il rotenone 5% addizionato di oli vegetali e lecitina di soia 

(tab.3, prodotto2) risulta più tossico per le uova. 

Variabili sono gli effetti degli altri formulati: solo l'assenzio agisce contro le uova del fitoseide e del fitofago, 

mentre la quassia riesce a controllare moderatamente le popolazioni dell'astigmata (tab. 3). 


Tabella 3. Estratti da vegetali vari: composizione e dose dei prodotti usati e loro effetto (E = tossicità globale, Abb. = 

mortalità secondo Abbott) su femmine e uova di due specie di acari di interesse agrario (T. urticae e N. californicus) e 

di una specie di acaro delle derrate (L. destructor). 



femmine 

E % 

uova 

Abb. % 

56 

femmine 

E % 

uova 

Abb. % 

femmine 

Abb. % 

uova 

Abb. % 

1 rotenone 5% 4g/l 46,92 32,04 57,69 42,85 78,30 26,09 


lecit. soia 

3 rotenone 6% 

(Rotena) 

4 riania 70% + oli 

veg. e lecit. soia 

4g+2g/l -3,85 93,33 99,66 62,33 44,32 34,78 

3cc/l ---- ---- 94,95 -1,27 ---- ---- 

4g+2g/l 6,47 40,03 -35,75 -3,89 24,85 -30,43 

5 assenzio 80% 8g/l 26,74 37,37 -3,84 70,13 2,04 -26,09 


lecit. soia 

8g2g/l 27,65 18,71 13,57 25,97 9,86 -30,43 

7 aglio 85% 1g/l -1,80 16,05 33,56 0,00 -7,05 -30,43 

8 n. 7+ oli veg. e 

lecit. soia 

1g+2g/l 29,08 33,38 -10,13 0,00 37,82 -26,09 

9 quassia 50% 5cc/l -35,41 -6,66 ---- ---- 45,34 47,83 

10 n. 9 + 

acidi grassi 30% 

5cc+4g/l 20,00 -2,66 ---- ---- 52,76 34,78 

CONSIDERAZIONI CONCLUSIVE 

I prodotti con lo stesso principio attivo miscelati o addizionati con differenti bagnanti ed emulsionanti 

determinano una diversa mortalità sulle specie considerate da cui si deduce che anche gli additivi possono condizionare 

l’entità delle risposte. 

Nell’ambito dei prodotti saggiati, in generale, si può osservare che quelli che esplicano azione acaricida sono 

più tossici per il predatore che per il fitofago del quale aumentano spesso la fecondità. Per gli acari delle piante, i 

risultati più promettenti si hanno essenzialmente con il prodotto contenente azadirachtina A a bassa concentrazione e 

olio di neem: questo formulato pur non essendo efficace sugli adulti del tetranichide, determina alta mortalità delle loro 

uova, e non incide invece sulla vitalità delle uova e delle femmine del fitoseide predatore, né sulla loro fecondità. 

Per l'acaro delle derrate, gli estratti di derris e quassia sono risultati i prodotti più tossici sugli stadi mobili 

mentre, sulle uova, gli estratti a base di piretro hanno determinato la più alta mortalità. 

RIASSUNTO 

I derivati da neem, derris, equiseto, riania, assenzio, aglio, crisantemo e quassia somministrati puri o con 

l’aggiunta di bagnanti od emulsionanti sono stati saggiati su Tetranychus urticae (specie fitofaga), Neoseiulus 


californicus (fitoseide predatore) e Lepidoglyphus destructor (astigmata comunemente conosciuto come acaro delle 

derrate alimentari) per valutare la loro tossicità sulle uova e le femmine e gli effetti sulla fecondità. 

Su tutte le specie i prodotti saggiati sono stati più efficaci sulle uova che sulle forme mobili. La tossicità più 

alta sulle femmine è stata evidenziata sul predatore. 

Solo i prodotti a base di azadiractina A a bassa concentrazione e olio di neem risultano efficaci sugli adulti e 

sulle uova del tetranichide incidendo poco sulla vitalità delle uova e delle femmine del fitoseide. Per l’acaro delle 

derrate gli estratti di derris e quassia sono risultati i più tossici. 

BIBLIOGRAFIA 

ABBOTT W.S., 1925 - A method of computing the effectiveness of an insecticide .- J. Econ. Entomol., 18: 265-267. 

CASTAGNOLI M., SIMONI S., GOGGIOLI D., 2000 - Attività biologica di sostanze vegetali nei confronti di Tetranychus 

urticae Koch (Acari Tetranichidae) e del suo predatore Neoseiulus californicus (McGregor)(Acari 

Phytoseiidae).- Redia, 83: 141-150. 

CASTAGNOLI M., ANGELI G., LIGUORI M., FORTI D., SIMONI S., 2002 - Side effects of botanical insecticides on 

predatory mite Amblyseius andersoni (Chant).- J. Pest Science 75: 122-127 

DENNEHY T.J., FURNHANNM A.W., DENHOLM I., 1993 - The microimmersion bioassay: a novel method for the topical 

application of pesticides to spider mites.- Pesticide Sci., 93: 47-54. 

HELLE W., OVERMEER W.P.J., 1985 - Toxicological test methods.- In: Spider mites, their biology, natural enemies and 

control. Vol 1A, Elsevier, Amsterdam, New York, Oxford, Tokyo: 391-395. 

GULATI R., MATHUR S., 1995 - Effect of Eucalyptus and Mentha leaves and Curcuma rhizomes on Tyrophagus 

putrescentiae (Scrank) (Acarina Acaridae) in wheat.- Experimental & Applied Acarology, 19: 511-518. 

MANSOUR F.A., ASCHER K.R.S., OMARI N.,1987 - Effects of neem (Azadirachta indica) seed kernel extracts from 

different solvents on the predacious mite Phytoseiulus persimilis and the phytophagous mite Tetranychus 

cinnabarinus. - Phytoparasitica, 15: 125-130. 

MANSOUR F.A., ASCHER K.R.S., ABO-MOCH F., 1997 - Effects of Neemgard on phytophagous and predacious mites 

and spiders. - Phytoparasitica, 25: 333-336. 

MOMEN F.M., REDA A.S., AMER S.A.A., 1997 - Effect of neem Azal-F on Tetranychus urticae and three predacious 

mites of the family Phytoseiidae.- Acta Phytopathol. Hungarica, 32: 355-362. 

NANNELLI R., SIMONI S., 2001 - Valutazione della tossicità di sostanze vegetali su femmine e uova di Lepidoglyphus 

destructor (Schrank) (Acari Glycyphagidae).- Redia, 84: 129-140. 

OVERMEER W.P.J., VAN ZOON A.Q., 1982 - A standardized method for testing the side effects of pesticides on the 

predacious mite Amblyseius potentillae (Acarina Phytoseiidae). - Entomophaga, 27: 357-364. 

PAPAIOANNOU-SOULIOTIS P., MARKOYANNAKI-PRINTZIOU D., ZOAKI-MALIOSSOVA D., 1997 - Side effects of Neemark 

(Azadirachta indica A. Juss ) and two vegetable oils formulations on Tetranychus urticae Koch and its predator 

Phytoseiulus persimilis Athias-Henriot.- Boll Zool. agr. Bachic. (ser II), 32: 25-33. 

PERRUCCI S., 1995 - Acaricidal activity of some essential oils and their constituents against Tyrophagus longior a mite 

of stored food.- Journal of Food Protection, 58 (5): 560-563. 

SCHMUTTERER H.,1990 - Properties and potential of natural pesticides from the neem tree, Azadirachta indica.- Annu. 

Rev. Entomol., 35: 271-297. 

SPOLLEN K.M., ISMAN M.B., 1996 - Acute and sublethal effects of a neem insecticide on the commercial biological 

control agents Phytoseiulus persimilis and Amblyseius cucumeris (Acari Phytoseiidae) and Aphidoletes 

aphidimyza (Diptera Cecidomyiidae).- J. Econ. Entomol., 89: 1379-1386. 

SUNDARAN K.M.S., SLOANE L., 1995 - Effects of pure and formulated azadirachtin, a neem-based biopesticides, on the 

phytophagous spider mite Tetranychus urticae Koch. - J. Environ. Sci. Health, B, Pesticides Food Contam. 

Agric. Wastes., 30: 811-814 

THIND B.B., MUGGLETON J., 1998 - A new bioassay for the detection of resistance to pesticides in the stored product 

mite Acarus siro (Acari Acaridae).- Experimental & Applied Acarology, 22: 543-552. 

TSOLAKIS H., LETO G., RAGUSA S., 1997 - Effects of some plant materials on Tetranychus urticae Koch (Acariformes 

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Conf. on pests in Agriculture, Montpellier 6-8 January, 1997, pp. 239-245. 


57

6 

CHANGES OF BIOCHEMICAL PARAMETERS IN SOIL AMENDED WITH MOIST OLIVE HUSKS 

Cristiano Casucci, Elga Monaci and Piero Perucci 

DIBIAGA, Ancona University, Via Brecce Bianche, 60131 Ancona, ITALY 

Key words: amendment, enzyme activity, soil fertility 

On the last twenty years the disposal of organic residues coming from agricultural crop and food industry is a very 

important problem relating to ecological risk, in particular in the middle Italy where the production of olive husks from 

oil-mill is very diffuse. Therefore, the use of olive husks like amendment can represent both a solution for problems 

connected to their disposal than a contribution to give an answer to the impoverishment of soils organic matter content 

by actual agricultural practices. The aim of this research was to evaluate the influence, both in laboratory and in field 

conditions, on some biochemical parameters correlated with soil fertility of the amendment with moist olive husks at 

different dose. 

Field experiments were carried out for a short-time (three years) in two soils amended with legal dose (D, 20 ton ha -1 

for year, equivalent to 1.47 g of organic C/kg of soil) and double dose (2D). Laboratory experiments in the same soils 

were carried out for one year and an additional dose of ten-fold field dose (10D) was employed. 

In field trials the biochemical parameters didn’t show significant changes in comparison with no-amended soils and, 

when present, their generally were positive. 

Concerning laboratory experiment the results were: 

• microbial biomass-C content: no-significant increases at the beginning of experiment for D and 2D but significant 

and constant increases during the entire period for 10D were found; 

• soil oxidation capacity: an inhibiting effect due to amendment was observed at the beginning of the trial which 

disappeared after the first month of incubation; 

• FDA-hydrolase capacity: an initial significant decrease at all doses, which disappeared after two month from 

amendment; 

• o-DPO activity: amendment at D and 2D didn’t cause significant changes, while a strong inhibition at 10D was 

observed. This tendency persisted during the early drawings; 

• β-glucosidase activity: the initial inhibiting effect (two months) was followed by an increase correlated with the 

organic amendment dose; 

• alkaline phosphatase activity: any influence was observed at D and 2D; 10D determined a significant decrease 

during the first two months, at the third month the activity value returned to the original values of controls. 


58

7 

NATURAL COMPOUNDS IN THE CONTROL OF POWDERY MILDEW ON CUCURBITS IN ORGANIC 

AGRICULTURE 

Fedele Casulli (1) , Agostino Santomauro (1) , Giuseppe Tauro (2) , Maria Antonia Gatto (1) , Franco Faretra (1) 

(1) Dipartimento Protezione delle Piante e Microbiologia Applicata, University of Bari, Italy 

(2) Centro di Ricerca e Sperimentazione in Agricoltura "Basile Caramia", Locorotondo (Bari), Italy 

In Italy, Cucurbitaceae are important vegetable crops grown especially in Central and Southern Italy (Sicily, Latium 

and Apulia). Melon (Cucumis melo L.), watermelon [Citrullus lanatus (Thunb) Matsum et Nakai], courgette (Cucurbita 

pepo L.) and cucumber (Cucumis sativus L.) are the prevalent species being cultivated on a surface of 21,400, 14,200, 

12,200 and 1,500 hectares, respectively (Istat, 2001). Powdery mildew, caused by Sphaerotheca fusca (Fr.) S. Blumer 

[syn. S. fuliginea (Schlecht. ex Fr). Poll.] is the most common and severe disease on the crops. Many fungicides are 

available for an effective control of the pathogen, but in view of their negative side effects, people look to more 

consumer- and environment-friendly alternatives, including natural compounds. The present paper reports the results of 

trials carried out under glasshouse and field conditions in order to evaluate the effectiveness of natural compounds in 

the control of powdery mildew in organic agriculture. 

Zucchini squash (Cucurbita pepo L., cv. Striato pugliese) and cucumber plants (C. sativus, cv. Mezzo lungo di 

Polignano) were grown individually in plastic pots (10 cm in diameter) in a glasshouse at 20-24°C and RH 70-80%, 

under daylight. Four-replicated plants, maintained at the two-three leaf stage by pruning were used for each thesis. The 

plants were artificially inoculated with S. fusca by spraying a suspension containing about 5⋅10 5 fresh conidia per ml. 

The tested compounds, including several natural substances allowed in organic agriculture (Table 1), were applied 

either 3 days before or 3 days after the artificial inoculation with the pathogen. Untreated check plants were sprayed 

only with water. Numbers and sizes of fungal colonies were assessed 11 days after inoculation on 10-cm 2 of the surface 

of each leaf. 

Natural substances were tested in field trials on melon (C. melo, cv. Barattiere) carried out during the summer. The 

statistical design was the complete randomised blocks with four replications and plots were made up of 8-10 plants in 

all trials. Spray schedules always were started before the appearance of first disease symptoms; treatments were 

repeated at one-week intervals. The severity of powdery mildew symptoms was periodically assessed evaluating the 

percentage of infected leaf surface. All data were submitted to variance analysis and mean values were separated by 

Duncan’s Multiple Range Test. 

According to the reduction of symptoms severity on treated plants as compared to the untreated check, the tested 

compounds were grouped in effective (higher than 70%), moderately effective (from 40 to 70%) or poorly effective 

(less than 40%) (Table 1). 

A good control of powdery mildew fungi was achieved in glasshouse as well as in field by fresh and dried milk (10%), 

which reduced infections over 90%. Among the other tested compounds, calcium and magnesium chloride, potassium, 

sodium or ammonium phosphate dibasic, pinolene, sodium bicarbonate and the mixtures of mineral oil with sodium 

bicarbonate or sodium silicate, significantly reduced the severity of S. fusca on leaves. These compounds showed the 

highest effectiveness when applied 3-4 days after the artificial inoculation of S. fusca. In the field, the same compounds 

yielded a satisfactory control of powdery mildew on the upper leaf surface of cucurbits, but they showed poor 

effectiveness against infections on stems and lower leaf surface, especially under high disease pressure. Mineral oil was 

effective when applied 3-4 days before artificial inoculation of S. fusca; this corroborate the finding that the compound 

acts also as inducer of defence mechanisms in plants. Ammonium phosphate monobasic, sodium silicate, sodium 

hydroxide, trisodium and tripotassium phosphate proved poorly effective at non-phytotoxic concentration while calcium 

carbonate, calcium sulphate, calcium silicate, sodium and potassium chloride, sodium and potassium phosphate 

monobasic were poorly effective against S. fusca. 


59

Table 1. Effectiveness against Sphaerotheca fusca of natural compounds tested in greenhouse and in field (a) . 

Compounds Rates Trials 

(%) Glasshouse Field 

Pinolene 1 +++ +++ 

Fresh milk 100 +++ 

Dried milk 10 +++ +++ 

Mineral oil 1 +++ +++ 

Sodium bicarbonate (NaHCO3) 0.5 +++ +++ 

Mineral oil + sodium bicarbonate 1+0.5 +++ +++ 

Mineral oil + sodium silicate 1+0.5 +++ +++ 

Potassium phosphate dibasic (K2HPO4) 0.5 +++ ++ 

Sodium phosphate dibasic (Na2HPO4) 0.5 +++ 

Ammonium phosphate dibasic [(NH4)2HPO4)] 1 +++ 

Calcium chloride (CaCl2) 0.5-1 +++ 

Magnesium chloride (MgCl2) 1 +++ 

Sodium silicate (Na2O . 2SiO2) 0.5 ++ 

Sodium hydroxide (NaOH) 0.25 ++ 

Ammonium phosphate monobasic (NH4H2PO4) 1 ++ 

Tripotassium phosphate (K3PO4) 0.5 ++ 

Trisodium phosphate (Na3PO4) 0.5 ++ 

Potassium phosphate monobasic (KH2PO4) 1 + 

Sodium phosphate monobasic (NaH2PO4) 0.5 + 

Potassium chloride (KCl) 1 + 

Sodium chloride (NaCl) 0.3-0.5 + 

Calcium carbonate (CaCO3) 1 + 

Calcium sulphate (CaSO4) 1 + 

Calcium silicate (CaO . 2SiO2) 1.5-2 + 

(a) +++ effective; ++ moderately effective; + poorly effective. 

The results herein discussed show that several compounds are effective in the early stages of infection or under low 

disease pressure, when applied repeatedly at short time intervals. Their effectiveness against powdery mildew proved to 

be influenced by temperature, time of application and especially by the uniformity of distribution on plants. In fact, field 

trials showed that one of their most important limits is the lack of control of powdery mildew on the lower surface of 

leaves. Cucurbits and other crops requiring sprays during a long harvesting time would benefit greatly from the usage 

of natural substances in crop protection. 

Acknowledgements: Work supported by the Italian Ministry of Agricultural and Forestry Policies in the frame of the 

research project “Technical tools for crop protection in organic agriculture” – Sub-project “Natural substances in crop 

protection from fungal diseases”. 


60

8 

METHODS FOR EVALUATION, IN CONTROLLED CONDITIONS, OF THE GROWTH OF OILSEED 

RAPE PROMOTED BY BACTERIA 

F. Citarrei, M. Scribano, S. Coranelli, C. Cellerino, L. Quattrocchi, L. Concezzi, and A. Ragni 


Tel: ++39 – 075 - 895091 Fax: ++39 – 075 – 888 776 

E-mail: aragni.bt@parco3a.org 

Abstract 

Growth promoters are microorganisms able to increase growth and productivity of plants by: atmospheric nitrogen 

fixation, mineral salts solubilization and siderophore synthesis (ferrous natural chelants), production of substances 

similar to plants hormones and with antagonistic effect towards phytopathogenic microorganisms. Growth promoters 

can influence the availability of nutritive elements or they can improve the phytosanitary conditions of the plants. In the 

present work we have tested the ability of some bacteria strain to promote the growth of oilseed rape [Brassica napus 

(DC) Metzger var. oleifera, Brassiceae] by using two types of experimentations: in vitro, in laboratory and in vivo, in 

greenhouse. The in vitro test was conducted with sterile seedlings monoxenically associated with bacteria isolates. In 

the in vivo trials, the seeds were treated with bacteria prior to sawing. Both methods are able to select and evaluate the 

promoting action of the bacteria towards the oilseed rape although the in vitro is faster and discriminate better the 

promoting effect of the bacteria. In vitro test 

Objective 

To test the ability of some bacteria strains to promote the rapeseed growth in vitro 

Materials and Methods 

Sterile rape seeds were sown on MS/2 culture media; sterile seedlings were transplanted in MS culture media inoculated 

with Pseudomonas sp. strain MK280 (Fig.1). This strain is able to produce siderophores that, as natural chelants, make 

iron available for plants. The treatments in the Pseudomonas test are reported in Table 1.The following growth 

parameters were checked at 15 and 30 days: plant height, plant fresh weight and plant dry weight. Table 1. 

Experimental conditions of rape seed in vitro test with Pseudomonas sp. MK280 

Codes Treatment description 

FeCl3+MK280 Ferric chloride + bacteria MK280 

FeCl3 

Ferric chloride without bacteria MK280 

Fe - Without iron, without bacteria MK280 

FeEDTA+MK280 Chelated iron + bacteria 

FeEDTA Chelated iron without bacteria MK280 

Figure 1. Sterile seedlings of oilseed rape rapes transplanted 

in MS culture media in a plant cell culture vessel. 


61

Figure 2. Oilseed rape plants grown in vitro with different treatments. 

For explication of codes, see Table 1. 

Fe - FeEDTA FeEDTA 

+ MK280 

FeCl3 

62 

FeCl3+MK280 


Figure 3. Effect of Pseudomonas sp. MK280 on rapeseed in vitro growth: plant height 

cm 

25 

20 

15 

10 

5 

0 

FeCl3 + MK280 FeCl3 FeEDTA + MK280 FeEDTA Fe - 

Treatments 

63 

plant height 15 days 


Figure 4. Effect of Pseudomonas sp. MK280 on rapeseed in vitro growth: plant fresh and dry weight 

Greenhouse trial 

g 

1,2 

1 

0,8 

0,6 

0,4 

0,2 

0 

FeCl3 + 

MK280 

FeCl3 FeEDTA + 

MK280 

Treatments 

fresh weight 15 days 

fresh weight 30 days 

dry weight 15 days 


FeEDTA Fe - 

Objective 

To test the ability of some bacteria strains to promote the rapeseed growth in controlled conditions. 


The trial was conducted using the following bacteria: Azospirillum brasilense (strain LMD78.36) or Pseudomonas sp. 

(strains MK280, Hv37a and AGS195B). The seeds were treated with bacteria strain cell suspensions; then the treated 

seeds were sown in a pot (one seed/pot) containing natural soil (Nitrogen= 0.08%; Organic matter =1.2%; Iron = 10.77 

mg kg -1 )The strains were tested on 20 plants, 10 plants were fertilized with Nitrogen (60 kg/ha) after 40 days from 

sowing; 10 were not fertilized. The following growth parameters were checked at 15 and 30 days: plant height, plant 

fresh weight and plant dry weight. 

Table 2. Colony Forming Unit from rape seeds treated with bacteria before sowing in greenhouse. 

Bacteria C.F.U/seed 

A. brasiliense LMD78.36 6 x 10 3 

Pseudomonas sp. MK280 1.2 x 10 5 

Pseudomonas sp. HV37a 6 x 10 5 


Pseudomonas sp. AGS195B 3 x 10 6 

Figure 5. Bacterial seed treatment effect on rapseed: plant height 

cm 

100 

80 

60 

40 

20 

0 

Control Hv37aR2 LMD78.36 AGS195B MK280 

Figure 6. Bacterial seed treatment effect on rapseed plant: dry weight 

g 

2,5 

2 

1,5 

1 

0,5 

0 

Treatments 

Results and Conclusions 

Results of the Pseudomonas sp. in vitro test were reported in Figures 2, 3 and 4. The strain MK280 promoted the 

rapeseed growth when a little available iron form (FeCl 3 ) is present in the culture media. Results of the greenhouse trial 

were reported in Figures 5 and 6. In the treatment without Nitrogen, after 80 days from sowing, the plants treated with 

Pseudomonas sp. strain MK280 showed a higher height and dry weight then control and plants treated by others 

bacteria. 

The present study showed that both methods were able to select and evaluate the promoting action of the bacteria 

towards the oilseed rape although the in vitro was faster and discriminated better the promoting effect of the bacteria. 


The study was funded by GAL Alto Tevere Valle delle Genti, Citta di Castello (Leader II project). 

Research work of F. Citarei, M. Scribano and C. Cellerino was supported by grant of 3A Parco Tecnologico 

Agroalimentare dell’Umbria, in the frame of Umbria Region Training programm (European Social Forum found). 

64 



plant height 80 days + N 

Control Hv37aR2 LMD78.36 AGS195B MK280 

Treatments 



dry weight 80 days + N 


9 

IMPROVEMENT OF SOIL PROPERTIES AND PHYTOPARASITIC NEMATODES SUPPRESSION BY 

COMPOSTED OLIVE MILL WASTE 

Convertini Grazia (1) , Ferri Donato (1) , Sasanelli Nicola (2) , D’Addabbo Trifone (2) 

(1) Istituto Sperimentale Agronomico (MIPAF) – Via Celso Ulpiani, 5 - 70125 Bari, Italy. E-mail: 

donatoferri.isaba@tuttopmi.it 

(2) Istituto per la Protezione delle Piante - Sez. di Bari - C.N.R. - Via Amendola 165/A, 70126 Bari, Italy. E -mail: 

nemans13@area.ba.cnr.it 

Summary. 

The effect of composted olive mill wastes (composted - OMW) soil amendments on phytoparasitic nematodes and on 

soil fertility was investigated in a sandy soil heavely infested by Meloidogyne incognita in southern Italy (Apulia 

region). Composted olive pomace obtained by mixing fresh solid cake coming from the three-phase decanter (for olive 

oil production) with farmyard manure (10, 20 and 40 t ha -1 ), another compost obtained by mixing exhausted solid 

cake (chemical extraction of residue olive oil) with wheat straw and poultry manure (10, 20 and 40 t ha -1 ) and raw 

sewage supplied at 40 and 80 m 3 ha -1 , were compared with two controls : i) untreated soil; ii) treatment with 

fenamiphos (traditional nematicide) at 0.3 t ha -1 . Tomato crop yield, soil nematode population and root gall index were 

recorded in all the plots. Soil fertility parameters, as total N, nitrates, ammonium, total, extracted and humified organic 

C, NaHCO3-P, exchangeable bases, heavy metals were determined for the most significant rate of each amendment. 

Crop yield was enhanced in all amended plots and pomace based composts were also suppressive on M. incognita. 

Moreover, compost amendments increased soil organic matter content, improved N availability and protected soil 

organic fractions from the fast decomposition occurring in the semi-arid conditions of southern Italy. 

Keywords: Control, Meloidogyne incognita, nematodes, olive mill wastes, soil fertility. 

INTRODUCTION 

The disposal of olive mill wastes (OMW), fresh pomace and raw sewage, represents a serious environmental 

problem in the areas of cultivation of olive, as large amounts of these materials are produced in a short period every 

year (Ranalli and De Mattia, 1996). 

The use of these materials as soil amendment could represent a possible alternative solution to this problem. Recent 

studies showed that in the soil characterized by poor fertility, OMW incorporation into the soil improves some chemical 

soil properties at different texture in southern Italy (Ferri et al., 2001). Also soil porosity, aggregates stability, 

hydrological properties were improved by the application of OMW (Pagliai, 1996), although contrasting results derived 

from previous experiments (Bonari and Ceccarini, 1993 ). 

Moreover, a suppressive action on soil phytoparasitic nematodes has been also reported among the effects of olive 

mill wastes incorporation into the soil (Rodriguez-Kabana et al., 1995; D’Addabbo and Sasanelli, 1996, Sasanelli et al., 

2002). 

Objective of the field experiment described in this paper was a comparative evaluation of the effect of fresh and 

exhausted composted olive pomace and raw sewage application on a population of the root-knot nematode 

Meloidogyne incognita and on the chemical properties of the soil. 

MATERIAL AND METHODS 

A sandy soil at Monteroni (province of Lecce, southern Italy), heavily infested by Meloidogyne incognita (Kofoid 

et White) Chitw. (Pi = 9 eggs and juveniles/cm 3 soil), was subdivided in 12 m 2 plots, spaced 1 m each other, according 

to a randomized block design with four replicates for each treatment. Composted olive pomace obtained by adding 

fresh solid cake (93%) from a three-phase decanter (for olive oil production) with farmyard manure (7 %) and a 

compost obtained by mixing exhausted pomace solid cake (91%) (after the chemical extraction of residue olive oil) with 

wheat straw (2%) and poultry manure (7 %) were distributed on the plot surface at 10, 20 and 40 t ha -1 rates and then 

incorporated into the soil at 25 – 30 cm depth by rotavation. Raw sewage was added at the dosages of 40 and 80 m 3 ha -1 . 

Untreated soil and 300 kg ha -1 fenamiphos applied before transplanting were used as controls. One month old seedlings 

of tomato (Lycopersicum esculentum L.), cv. Tondino di Zagaria, were transplanted into the plots three months after the 

treatments. During the growing season the field received the usual cultural practices. 

At the end of the crop cycle in each plot the tomato yield was recorded, the nematode gall infestation index on the 

roots was estimated on a 0 – 5 scale (0 for not galled roots and 5 for roots completely deformed by many large galls) 

(Di Vito et al., 1979) and the nematode population density was determined in the soil (Coolen, 1979). 


65

Soil samples were collected in the plots treated with : i) no input (CONTR); ii) fenamiphos (300 kg ha -1 ) 

(F.MPHOS); iii) raw sewage (80 mc ha -1 ) (R.S.); iv) exhausted pomace solid cake compost ( 20 t ha -1 ) (EOPC); v) fresh 

pomace solid cake compost (20 t ha -1 ) (FOPC), after the treatments and harvesting to evaluate soil N-NO3, N-NH4 

exchangeable, NaHCO3 - P (Olsen method), exchangeable bases, CEC, heavy metals concentration , total organic 

carbon (TOC), total extracted (TEC) and humified carbon (humic and fulvic acids, C HA + C FA ) (Sequi et al., 1986; 

Italian Ministry of Agricultural Resources, 1994). The degree of humification, DH% = (C HA + C FA )/TEC*100, the 

humification rate, HR% = (C HA + C FA )/TOC*100, and the humification index, HI= NH / (C HA + C FA ), were also 

calculated. 

Data were statistically analysed by the analysis of variance and means compared by Duncan’s Multiple Range Test 

(P=0.05). 

RESULTS AND DISCUSSION 

In the field experiment all the composts significantly increased the tomato yield compared to the untreated control, 

but were statistically lower than chemical treatment (Table 1). 

All the treatments significantly reduced the root infestation index and, with the exception of the lowest rates of raw 

sewage and exhausted pomace compost, the soil nematode population (Table 1). 

Results from this experiment and their comparison with those from the previous field experiments evidenciated that 

incorporation of OMW into the soil could result in a suppression, but not in a complete eradication of phytoparasitic 

nematode populations and, at low initial soil infestation, also in a crop yield increase. Therefore, the aim of soil 

amendments with these materials should be a progressive reduction of infestation level under the tolerance limit of the 

target nematode species (Sasanelli, 1994). 

Table 1 - Effects of different olive mill wastes amendments on Meloidogyne incognita on tomato (cv. Tondino di 

Zagaria). 

Treatment 

Dose 

(t x 

ha -1 ) 

66 

Tomato 

yield 

(t x ha -1 ) 

Root gall index 

(0 – 5) 

Eggs and 

juveniles/cm 3 

soil 

Untreated control - 25.4 a 4,5 b 19.8 d 

Fenamiphos 0,3 40.5 e 2,7 a 10.6 abc 

Raw sewage 4 32.3 bc 2,4 a 15.1 cd 

8 29.0 b 2,5 a 5.2 a 

Exhausted olive pomace compost 10 36.8 d 2,1 a 13.7 bcd 

20 36.6 d 2,2 a 9.2 abc 

40 36.3 d 2,2 a 11.2 abc 

Fresh olive pomace compost 10 32.1 bc 2,8 a 5.3 a 

20 33.7 cd 2,2 a 7.1 ab 

40 35.8 cd 2,6 a 9.3 abc 

Data are means of four replications; data followed by the same letter on the same column are not significantly 

different according to Duncan’s Multiple Range Test (P = 0.01). 

In the Figure 1 A can be observed a poor increasing trend of soil N-NO3 contents respectively for “R.S.”, 

“EOPC”, “FOPC” treatments and lowest N-NO3 content in “fenamiphos-treated” plots. 

Soil NaHCO3-P content was unaffected from experimental treatments, but soil exchangeable K showed higher 

value in "fenamiphos-treated" plots (Figure 1A). On the contrary, in “FOPC-treated” plots higher “exchangeable-Ca” 

content was recorded , while similar trends were observed in exchangeable Ca in “fenamiphos-”, “R.S.-”, “EOPC- 

treated” plots. No differences were observed on exchangeable Na and Mg among treated plots compared with the 

“control” (Figure 1 B ). 

Among the heavy metals of the soil (Figure 1 C ), only Zn content was higher in “EOPC-“ and “FOPC-treated” 

plots probably because during the composting processes of solid residual cake of olive milling ( fresh or exhausted ) a 

Zn concentration could be verified. 

Soil organic C (Figure 1 D) contents (total and extracted) seem lower in the plots treated with OMW (raw or 

composted) in comparison to other treatments, because the incorporation into the soil of fresh organic matter improves 

biological activity (Ocio et al., 1991). No variations were recorded on humified organic C, probably for the short period 

of trial. 


(mg kg-1) 

(mg kg-1) 

140 

120 

100 

80 

60 

40 

20 

0 

120 

100 

80 

60 

40 

20 

0 

b 

N-NO3 NaHCO3-P Exch.-K 

b 

n.s. n.s. 

b 

a 

Soil available nutrients 

a 

n.s. 

Zn Cu Pb 

Soil heavy metals 

b 

a 

b b b 

n.s. 

CONTR. 

F.MPHOS 

Soil properties reported in the Table 2 show that experimental treatments play a similar role on pH and 

CEC. The degree of humification (DH) results significantly higher in “R.S.-treated” plots than in 

“control” plots; on the other hand in “fenamiphos-” and “FOPC-treated” plots this parameter appears very lower 

than in “control” plots. Humification rate 

(HR) in all treated plots is lower than control. Finally humification index (HI) of “fenamiphos-“ and “FOPC-“ treated 

plots is higher than other plots similarly to the findings of degree of humification (DH). 

Table 2 - Variations of some soil properties as affected by experimental treatments. 

A 

R.S. 

EOPC 

FOPC 

CONTR. 

F.MPHOS 

R.S. 

EOPC 

FOPC 

Treatments pH CEC 

Humification parameters 

(meq/100 g) DH (%) HR (%) HI 

CONTR 8.30 9.55 20.4 b (1) 9.1 a 3.9 b 

F.MPHOS 8.32 10.13 16.0 c 7.4 c 5.2 a 

R.S. 8.20 9.81 22.7 a 8.1 b 3.4 b 

EOPC 8.29 9.06 21.3 b 8.1 b 3.7 b 

FOPC 8.23 9.29 16.3 c 6.1 c 5.1 a 

(1 ) – Values followed by the same letter are not significantly different at P < 0.05 

according to Duncan’s Multiple Range Test. 

In conclusion OMW soil amendments could be suggested for integrated nematode management strategies, as it 

provides a good pest suppressivity. 

After the first year of trial is very hard to record soil fertility variations as affected by experimental treatments. The 

experimental data show that both OMW soil amendments and traditional nematicide (fenamiphos) don’t determine 

decrease of soil available nutrients, soil exchangeable bases. On the other hand the effects on soil heavy metals (Zn, 

Cu, Pb) are unsignificant, when were compared all the treatments with “control”. Very interesting trends were observed 

on soil organic C and humification parameters. The apparent increase observed on soil organic C (TOC, TEC) in the 

plots untreated (CONTR) and treated with fenamiphos (F.MPHOS) seems in contrast with a light increase of 

67 

(mg kg-1) 

2500 

2000 

1500 

1000 

5 

4 

3 

2 

1 

0 

500 

0 

Ca Mg Na 

Soil exchangeable bases 

CONTR. 

F.MPHOS 

R.S. 

EOPC 

FOPC 

C 7 

a 

D 

6 

a 

b b b 

( g kg-1 ) 

a ab 

b 

b 

ab 

n.s. 

Total Extract. Humified 

Soil organic Carbon 

Figure 1 – Soil fertility variations as affected by experimental treatments. 

(For each parameter columns followed by different letters are significanty different at P

humification parameters recorded in the other plots (R.S.-, EOPC-, FOPC-treated). Probably, the incorporation into the 

soil of OMW (raw or composted) determines an improvement of soil o.m. transformations. 

Moreover it is necessary to continue experimental research to verify when OMW and traditional nematicide effects 

are evident and eventually different on soil fertility dynamics. 

Work carried out in the frame-work of the O.P. CNR-MURST, Law 95/95, National Research Program: “Reflui Agro- 

Industriali – Sottoprogetto Reflui Oleari”. 

REFERENCES 

Bonari E. e L. Ceccarini, 1993. Sugli effetti dello spargimento delle acque di vegetazione sul terreno agrario: risultati 

di una ricerca sperimentale. Genio Rurale, 5: pp.60-67 

Coolen W. A., 1979. Methods for the extraction of Meloidogyne spp. and other nematodes from roots and soil. In: 

Root-knot nematodes (Meloidogyne species) Systematics, Biology and Control. (F. Lamberti and C.E. Taylor Eds), 

Academic Press, London, UK, pp. 317-329. 

D’Addabbo T. and N. Sasanelli, 1996. Effect of olive pomace soil amendment on Meloidogyne incognita. 

Nematologia mediterranea, 24, pp. 91-94. 

Di Vito M., F. Lamberti e A. Carella. 1979. La resistenza del pomodoro nei confronti dei nematodi galligeni: 

prospettive e possibilità. Rivista di Agronomia 13: pp. 313-322. 

Ferri, D., G. Convertini, F. Montemurro e C. Vitti, 2001. Evoluzione di alcune proprietà chimiche di terreni pugliesi 

ammendati con reflui oleari. Atti Giornata di Studio “ Prospettive di utilizzzione agronomica dei reflui oleari” ( Bari, 14 

febbraio). 

MIRAAF, 1994. Metodi ufficiali di analisi chimica del suolo. 

Ocio J.A., P.C. Brookes and D.S. Jenkinson, 1991. Field incorporation of straw and its effects on soil microbial 

biomass and soil inorganic N. Soil Biol. Biochem. 23: pp.171-176. 

Pagliai M., 1996. Effetti della somministrazione di acque reflue di frantoi oleari sulle caratteristiche fisiche del suolo. 

Atti Convegno Int. su “Trattamento e riciclaggio in agricoltura dei sottoprodotti dell’industria olearia”. Lecce, 8-9 

marzo. 

Ranalli A. and G. De Mattia. 1996. Role of piloted biological processes in the purification of oil mill waste water. 

Riv. Ital. Sost. Grasse, LXXIII: pp.61-66. 

Rodriguez-Kabana R., V. Estaun, R.J. Pinochet and O. Marfa’, 1995. Mixtures of olive pomace with different 

nitrogen sources for the control of Meloidogyne spp. on tomato. Journal of Nematology, 27 (4S), pp. 575 -584. 

Sasanelli N. 1994. Tables of Nematode-Pathogenicity. Nematologia mediterranea, 22: p.153-157. 

Sasanelli N., T. D’Addabbo, G. Convertini and D. Ferri, 2002. Soil Phytoparasitic Nematodes Suppression and 

Changes of Chemical Properties Determined by Waste Residues from Olive Oil Extraction. Proceedings of 12 th ISCO 

Conference, May 26-31, 2002 Beijing China. Vol. III: pp. 588-592. 

Sequi, P., M. De Nobili, L. Leita and G. Cercignani, 1986. A new index of humification. Agrochimica, 30: pp.175 – 

179 


68

10 

NEMATICIDAL ACTIVITY OF AQUEOUS EXTRACTS FROM RUE (RUTA GRAVEOLENS L.) 

T. D’Addabbo and N. Sasanelli 

Istituto per la Protezione delle Piante- Cnr, Sezione di Bari 

Via G. Amendola 165/A – 70126 Bari. Italy 

E-mail: nematd12@area.ba.cnr.it 

Summary. The effect of leaf and root aqueous crude extracts and of root leachates of R. graveolens on the root-knot 

nematodes Meloidogine arenaria, M. hapla, M. incognita and M. javanica, and on the beet cyst nematode Heterodera 

schachtii was investigated in a in vitro experiment. Aqueous extracts were prepared by soaking green leaves or roots in 

distilled water and root leachates were collected by drenching the rue cultivated soil with excess tap water. Egg masses 

of each Meloidogyne species and cysts of H. schachtii were incubated in the test solutions over a four weeks period. 

Immersion in rue extracts significantly suppressed juvenile emergence from the egg masses of all the four 

Meloidogyne species and from cysts of H. schachtii. Leaf extract was always more effective than root extract for 

Meloidogyne species, whereas no difference resulted in H. schachtii. No synergic effect derived from the combination 

of the two extracts. No significant emergence reduction resulted from the immersion in rue root leachates for any of the 

tested nematode species. 

INTRODUCTION 

Several plants are reported to minimize nematode damage in vegetable and field crops by producing nematicidal 

(killing) and nematistatic (suppressive) organic compounds. These natural compounds can be released from the roots of 

living plants or by plant tissues incorporated into the soil as a green manure (Grainge and Ahmed, 1988). 

The biocidal effect of extracts from rue (Ruta graveolens L.) was previously reported on insects, fungi and weeds 

(Aliotta et al., 1994; 1999; Oliva et al., 1999), but few information is available on the effect on phytoparasitic 

nematodes. Therefore an experiment was undertaken to investigate the in vitro effect of leaf and root aqueous extracts 

and of root leachates of R. graveolens on four different species of root-knot nematodes, Meloidogine arenaria (Neal) 

Chitw., M. hapla Chitw., M. incognita (Kofoid et White) Chitw.and M. javanica (Treub) Chitw. and on the beet cyst 

nematode Heterodera schachtii Schmidt. 

MATERIALS AND METHODS 

Aqueous extracts of rue were prepared by soaking 100 g green leaves or roots, or 50 g when in combination, in 400 ml 

distilled water for 24 hrs. Tissues were then macerated in a blender and the suspension filtered through filter paper. 

Root leachates were collected from three month old plants, cultivated in fifteen 2,500 cm 3 clay pots, by drenching the 

soil with excess tap water. The leachates were then centrifuged at 1,300 g for 30 min, stored in plastic bottles, and kept 

in a freezer until required. In the experiment on H. schachtii each plant extract or root leachate was adjusted to 3 mM 

adding an equal volume of 6 mM of zinc sulphate (Clarke and Shepherd, 1966). 

The four Meloidogyne populations were reared on tomato cv. Roma in a glasshouse at 20-25 °C, whereas H. schachtii 

was collected from an infested field at Avezzano (L’Aquila). Batches of twenty egg masses (averaging 20,000 eggs per 

mass) of each Meloidogyne species or 100 cysts of H. schachtii (123 egg/cyst) were placed on 2 cm diam sieves (215 

µm aperture) and each sieve in a 3.5 cm diam Petri dish. Three ml of each test solution, were then added to four batches 

of egg masses or cysts. Distilled water (for Meloidogyne species) or 3 mM zinc sulphate solution (for H. schachtii) and 

a 5 µg/ml aqueous solution of fenamiphos were used as controls. 

The dishes were arranged in a complete randomized block design with four replicates of each treatment: egg masses of 

Meloidogyne species were incubated at 25 °C, whereas the cysts of H. schachtii were mantained at 20 °C. Emerging 

juveniles were removed and counted at weekly intervals, renewing the hatching solutions at the same time, over a nine 

or ten weeks period, respectively for Meloidogyne species and H. schachtii. Egg masses and cysts were removed from 

the test solutions after the first four weeks and the incubation continued in distilled water or, for cysts of H. schachtii, in 

the zinc sulphate aqueous solution. 

At the end of the experiment the egg masses were immersed in a 1% sodium hypochlorite aqueous solution (Hussey and 

Barker, 1973), whereas the cysts were crushed, according to Seinhorst and Den Ouden (1966). Unhatched eggs from 

egg masses and cysts were then counted. Numbers of juveniles emerging weekly were expressed as cumulative percent 

of the total initial population. Data were statistically analysed and compared by LSD’s test. 


69


Emergence of juveniles from the egg masses immersed in leaf and root extracts of Ruta graveolens, either alone or 

combined, was significantly suppressed compared to the water control in all the four Meloidogyne species (Table 1). 

Leaf extract was always more effective than root extract and resulted statistically more suppressive also than 

fenamiphos solution. No synergic effect was derived from the combination of the two extracts, as final hatch was higher 

than in leaf extract alone. Egg masses immersed in root leachates gave a final hatch similar to control. 

The ultimate egg hatch of H. schachtii cysts incubated in single root and leaf extracts was significantly less than in 3 

mM zinc sulphate and fenamiphos (Fig. 1). Suppressivity of the extracts decreased when in combination, as no 

difference there was in final hatch compared to the control. There was no difference between leaf and root extracts, 

whereas their combination resulted in a significantly higher egg hatch. As found in Meloidogyne species, no significant 

emergence reduction was found for the root leachates. 

In conclusion, the experiment evidenciated the nematicidal properties of rue extracts. The nematicidal compounds 

seemed to be present in all the plant tissues, but especially in leaves. A possible antagonistic effect of leaf and root 

extracts emerged from the results, that could be attributable to a negative interaction of the active principles responsible 

for the suppressive effect. The absence of a nematicidal effect in root leachates indicated that nematicidal compounds 

are present only in plant tissues, but not released into the soil. 

LITERATURE CITED 

Aliotta, G., and G. Cafiero, (1994). Potential allelochemicals from Ruta graveolens L. and their action on radish seeds. 

Journal of Chemical Ecology 20 (11): 2761-2775. 

Clarke A.J. and A.M. Shepherd, (1966). Inorganic ions and the hatching of Heterodera spp. Annals of Applied 

Biology 58: 49–508. 

Grainge M. and S. Ahmed, (1988). Handbook of Plants with Pest-Control Properties. (J. Wiley and Sons eds.) New 

York, pp. 238-248. 

Hussey R.S. and K.R. Barker, (1973). A comparison of methods of collecting inocula of Meloidogyne spp. including a 

new technique. Plant Disease Reporter 57: 1025-1028. 

Landolt, P. J.,R. W. Hofstetter and L. L. Biddick , (1999). Plant essential oils as arrestants and repellents for 

neonate larvae of the codling moth (Lepidoptera: Tortricidae). Environmental Entomology 28 (6): 954-960. 

Oliva, A. and E. Lahoz, (1999). Fungistatic activity of Ruta graveolens extract and its allelochemicals. Journal of 

Chemical Ecology 25 (3): 519-526. 

Seinhorst J.W. and H. Den Ouden (1966). An improvement of Bijloo’s method for determining the egg content of 

Heterodera cysts. Nematologica (12): 170-171. 

Table 1. Effect of root and leaf extracts and root leachates of rue (Ruta graveolens L.) on the total percent cumulative 

hatch of four different Meloidogyne species. 

Treatments 

70 

Meloidogyne species 

M. arenaria M. hapla M. incognita M. javanica 

Leaf extract 12 6 12 3 

Root extract 59 43 61 23 

Root and leaf extract 20 21 49 21 

Root leachates 83 91 80 89 

Fenamiphos 38 47 29 31 

Distilled water 81 87 75 92 

L.S.D. 0.05 11 12 15 15 

L.S.D. 0.01 15 15 20 20 


% cumulative hatch 

40 

35 

30 

25 

20 

15 

10 

5 

0 

LE RE LRE LCH FEN CTRL 0,05 0,01 

Fig. 1. Effect of rue (Ruta graveolens L.) extracts on the percent cumulative hatch of Heterodera schachtii (LE = leaf 

extract; RE = root extract; LRE = leaf + root extract; LCH = root leachates; FEN = 5 µg/ml fenamiphos aqueous 

solution; CTRL = 3 mM zinc sulphate solution). 


71

11 

VALUTAZIONE DELL’EFFICACIA INSETTICIDA DI RYANIA, ROTENONE, AZADIRACTINA E 

BEAUVERIA BASSIANA 

Elisabetta Gargani, Giovanna Del Bene 

Istituto Sperimentale per la Zoologia Agraria – Via Lanciola, 12 A - 50125 Firenze. 

e-mail: giovannadelbene@virgilio.it 

Nell’ambito del P.F. MiPAF “Difesa delle produzioni in agricoltura biologica”, l’ISZA, sezione di Entomologia 

Agraria, ha effettuato prove sperimentali sull’efficacia insetticida di prodotti di origine naturale (Del Bene et al., 2000): 

ryania, rotenone, azadiractina e Beauveria bassiana. Le prove sono state condotte in laboratorio, in serra e in campo su 

varie specie vegetali di interesse ortoflorovivaistico, infestate sia naturalmente sia artificialmente da insetti appartenenti 

agli ordini dei Tisanotteri (Tripidi Heliothrips haemorrhoidalis e Frankliniella occidentalis), Rincoti (Triozide 

Lauritrioza alacris e Aleurodide Trialeurodes vaporariorum), Lepidotteri (Gracillariide Phyllocnistis citrella) e 

Coleotteri (Crisomelide Pyrrhalta viburni). Sono stati inoltre studiati gli effetti collaterali nei confronti del parassitoide 

Imenottero Afelinide Encarsia formosa. 

I prodotti sono stati sperimentati in formulazioni e dosaggi diversi con una sola applicazione o 2-3 applicazioni a 

distanza di 7 giorni. I controlli sono stati effettuati di norma a cadenza settimanale, conteggiando gli individui vivi e 

morti, distinti per stadio di sviluppo. Sono state calcolate le percentuali di sopravvivenza e di mortalità (M) secondo 

Abbott. 

RYANIA 

Il principio attivo, costituito dagli alcaloidi ryanodine, è estratto da Ryania speciosa, arbusto della famiglia 

Flacourtiaceae, nativo di Trinidad e del Bacino dell’Amazzonia. Le ryanodine agiscono soprattutto per ingestione e 

hanno una persistenza di due settimane; sono tossiche per i mammiferi (Tremblay, 1985; Casida et al., 1987). 

Sono stati impiegati 2 formulati sperimentali, Tigosan liquido (3-6 ml/l) e Tigosan polvere (3-5 g/l), di cui solo quello 

in polvere ha dimostrato una certa efficacia (tab. 1). 

Tab. 1 – Percentuali di mortalità (sec. Abbott) ottenute con Ryania. 

Formulato Dose ml/l Insetto Stadio Pianta % controllo 

Tigosan L 3 H. haemorrhoidalis Nean. - adulti viburno - labor. 7,5 

6 " " 22 

Tigosan WP 3 mirto - campo 22 

Tigosan WP 5 F. occidentalis Nean. - adulti pomodoro - serra 55,6 

5 fagiolino - labor. 40 

Tigosan WP 5 L. alacris Ninfe I-II alloro - labor. 45,2 

Ninfe III-V " " 10,5 

Tigosan WP 5 – 2tr. T. vaporariorum * Neanidi I pomodoro - serra 11,5 

5 – 2tr. Nean. II-IV " " 10,4 

Tigosan WP 5 P. citrella Larve I-III agrumi - vivaio 0 

Tigosan WP 5 P. viburni Larve I viburno - labor. 100 

Larve II-III " " 53,3 

Larve I-II viburno - serra 82,5 

Adulti viburno - labor. 66,7 

Adulti viburno - campo 0 

* Nessun effetto su E. formosa (100% sfarfallamento) 

Ryania ha dato esiti variabili contro P. viburni: in laboratorio la mortalità delle larve di I età ha raggiunto il 100 % con 

la dose di 5 g/l, mentre quella delle larve di età superiore è stata del 55,3% e quella degli adulti del 66,7%; in serra è 

stata confermata l’efficacia sulle larve I-II (82,5 % M) mentre in campo non è stato registrato alcun controllo degli 

adulti. E’ risultata mediamente efficace nei confronti di F. occidentalis su pomodoro in serra (55,6%M) e selettiva nei 

confronti di E. formosa. 


72

ROTENONE 

Il principio attivo è estratto dalle radici di Derris elliptica e di altre Papillionaceae appartenenti ai generi Lonchocarpus 

e Tephrosia; agisce per contatto e ingestione (La Torre et al., 2002). Ha bassa persistenza e si decompone rapidamente 

con l’esposizione all’aria e alla luce; non è selettivo nei confronti degli insetti utili né dei fitoseidi (Tsolakis et al., 1997) 

ed è tossico per i vertebrati (Tremblay, 1985). In Italia il prodotto è stato registrato come liquido emulsionabile ad 

ampio spettro di azione, alla concentrazione del 6% di p.a. (“Rotena” Serbios, Tossico) e al la concentrazione del 4% 

(“Bioroten” Intrachem, Nocivo). 

Sono stati impiegati 3 formulati: Derisan (non registrato, 3-6 ml/l); Rotena (2,5 ml/l); Bioroten (3 ml/l) (tab. 2). 

Tab. 2 – Percentuali di mortalità (sec. Abbott) ottenute con Rotenone. 


Derisan 6 H . haemorrhoidalis Nean. - adulti viburno - labor. 50 

Rotena 2,5 – 2tr. viburno - campo 0 

Derisan 3 F. occidentalis Nean. - adulti pomodoro - serra 0 

fagiolino - labor. 13,4 

Bioroten 3 – 3tr. limonium - serra 49,3 

Derisan 6 E. phyllireae NinfeIV-V fillirea - labor. 0 

Rotena 2,5 L. alacris Ninfe I-II alloro - labor. 93,1 


Rotena 2,5 – 3tr. T. vaporariorum* Nean. I pomodoro -serra 55,7 

Nean. II-IV " " 60 

Rotena 2,5 P. citrella Larve I-II agrumi - vivaio 0 

Derisan 3 P. viburni Larve I viburno - labor. 7,9 

Bioroten 3 Larve I-II viburno - serra 100 

Rotena 2,5 Adulti viburno - labor. 93,3 

*Effetti negativi su E. formosa (sfarfallamento da 8, 75% a 32,5%) 

Rotenone è risultato mediamente efficace nei confronti di H. haemorrhoidalis con mortalità del 50% (Derisan) e 

verso le neanidi di T. vaporariorum con 55,7 – 60% M (Rotena); assai efficace verso le giovani ninfe di L. alacris con 

mortalità del 93,1% (Rotena). Nei confronti di P. viburni, la resa del prodotto è stata assai diversa a seconda dello 

stadio trattato e del formulato usato. Infatti mentre le larve di I età, stadio più vulnerabile, sono sopravvissute in 

laboratorio al trattamento con Derisan, la mortalità di larve I-II trattate con Bioroten è stata del 100% e quella degli 

adulti trattati con Rotena è stata del 93,3%. Rotena ha avuto comunque effetti collaterali negativi molto pesanti su E. 

formosa, le cui percentuali di sfarfallamento sono state ridotte significativamente. 

AZADIRACTINA 

Dall’albero Azadirachta indica (neem), della famiglia Meliaceae, originario dell’India, viene 

estratto un olio contenente numerosi principi attivi appartenenti alla classe dei limonoidi (azadiractine), con proprietà 

antiparassitarie (Schmutterer, 1990; La Torre et al., 2002). L’estratto ha azione come regolatore di crescita, 

fagodeterrente e repellente e riduce la fecondità degli adulti e la vitalità delle uova (Rovesti e Deseo, 1990; Bezzi e 

Caden, 1991; Capella et al., 2000); agisce per ingestione e contatto e presenta un elevato potere di penetrazione (Isman 

et al., 1991). In Italia è commerciato come biostimolante vegetale (“Stardoor” Intrachem, EC 4,5%); inoltre sono stati 

registrati come insetticidi due prodotti a base di azadiractina A, che rappresenta la componente con le più elevate 

proprietà insetticide (“Oikos” Sipcam e “Diractin” Serbios, EC 3%, non classificati tossicologicamente). 

I formulati impiegati sono stati 4: Olnisan (non registrato, 2,5 ml/l); Stardoor (0,7 ml/l); Oikos (1-1,5 ml/l); Diractin 

(1,5 ml/l) (tab. 3). 


73

Tab. 3 – Percentuali di mortalità (sec. Abbott) ottenute con Azadiractina. 


Oikos 1,5 – 2tr. H. haemorrhoidalis Nean. - adulti viburno - campo 90,7 

Olnisan 2,5 F. occidentalis Nean. - adulti pomodoro - serra 9,4 

fagiolino - labor. 34 

Oikos 1,5 – 3tr. limonium - serra 89,9 

Diractin 1,5 – 3tr. " " 86,6 

Stardoor 0.7 L. alacris Ninfe I-II alloro - labor. 89,4 


Olnisan 2,5 Ninfe I-V alloro - vivaio 29,5 

Stardoor 0,7 T. vaporariorum* Nean. I pomodoro- serra 18,7 

Olnisan 2,5 P. citrella Larve I-II agrumi - vivaio 18 

Oikos 1,5 – 2tr. " " 48,1 

Diractin 1,5 – 2tr. " " 55,6 

Olnisan 2,5 P. viburni Larve I viburno - labor. 10,5 

Diractin 1,5 – 3tr. Larve I-II viburno - serra 90,6 

Stardoor 0,7 Adulti viburno - labor. 13,3 

Oikos 1 – 2tr. Adulti viburno - campo 0 

*Effetti negativi su E. formosa (sfarfallamento 17,5%) 

I diversi formulati, escluso Olnisan, hanno dato risultati positivi nei confronti delle ninfe di I e II età di L. alacris 

(Stardoor 89,4 % M), nei confronti di F. occidentalis (Oikos 89,9% M, Diractin 86,6% M) e H. haemorrhoidalis (Oikos 

90,7% M); hanno mostrato un ottimo contenimento di larve I-II di P. viburni (Diractin 90,6% M) e una certa efficacia 

contro larve I-II di P. citrella (Oikos 48,15% M, Diractin 55,6% M). Gli effetti collaterali nei confronti di E. formosa 

sono stati negativi con Stardoor . 

BEAUVERIA BASSIANA 

E’ un Deuteromicete agente del “calcinaccio” del baco da seta, scoperto nel 1835 da Agostino Bassi, che ne ipotizzò 

per primo l’uso nella lotta agli insetti dannosi. Le spore del fungo, a contatto con l’insetto, germinano penetrando 

attraverso la cuticola, mediante una combinazione di azioni meccaniche ed enzimatiche: lo sviluppo successivo del 

fungo, accompagnato da produzione di tossine, determina la morte dell’insetto solitamente in 3-5 giorni (Magnano di 

San Lio e Vacante, 1989; Benuzzi e Santopolo, 2001; La Torre et al., 2002). B. bassiana ha ampio spettro di attività e 

agisce per contatto in ambiente umido. Come bioinsetticida è stato prodotto a partire da un ceppo isolato su 

Anthonomus grandis: il formulato registrato in Italia è “Naturalis” (Intrachem, Irritante), sospensione concentrata al 

7,16% di B. bassiana, contenente 2,3 x 10 7 conidiospore/ml (tab. 4). 

Tab 4 – Percentuali di mortalità (sec. Abbott) ottenute con B. bassiana. 


Naturalis 1 – 2tr. H. haemorrhoidalis Nean. - adulti viburno - campo 41,3 

1,5 – 3tr. F. occidentalis Nean. - adulti rosa - serra 81 

limonium - serra 85 

1 P. citrella Larve I-II agrumi - vivaio 0 

1,5 – 3tr. P. viburni Larve I-II viburno - serra 81,1 

1– 3tr. Adulti viburno - labor. 55,3 

1 – 2tr. viburno - campo 3,4 

Naturalis (1-1,5 ml/l), a seguito di trattamenti ripetuti, ha assicurato un controllo soddisfacente in serra di F. 

occidentalis (81 –85 % M) e di larve I-II di P. viburni (81,1% M), ma non di H. haemorrhoidalis in campo (41,35 % 

M), né di adulti di P. viburni in laboratorio (55,3% M) e in campo (3,4% M). 

Riassumendo, i prodotti biologici testati hanno dato risultati variabili a seconda dell’impiego in laboratorio o campo, 

in base alla diversa formulazione e dose, alla specie di insetto e alla sua fase di sviluppo al momento del trattamento. 

Risultati chiaramente positivi (controllo > 90%) sono stati ottenuti con: 

- ryania contro larve I di P. viburni (Tigosan WP: 100% M.); 

- rotenone nei confronti di P. viburni (adulti: Rotena: 93,3% M , larve I-II: Bioroten: 100% M) e di L. alacris (ninfe I- 

II: Rotena: 93,1% M); 

- azadiractina contro H. haemorrhoidalis (Oikos: 90,7% M.) e larve I-II di P. viburni (Diractin: 90,6% M). 

Hanno comunque garantito un controllo superiore all’ 80%: 

- ryania nei confronti di larve I-II di P. viburni (Tigosan WP: 82,5% M); 


74

- azadiractina nei confronti di F. occidentalis ( Oikos: 89,9% M, Diractin: 86,6% M), ninfe I-II di L. alacris (Stardoor: 

89,4% M); 

- B. bassiana nei confronti di F. occidentalis (81% M su rosa, 85,05 % M su limonium) e di P. viburni (larve I-II 81,1 

% M). 

Nei confronti di E. formosa si sono avuti effetti collaterali negativi con Rotena e Stardoor, mentre ryania è risultata 

selettiva. 

Bibliografia 

BENUZZI M., SANTOPOLO F., 2001 – Naturalis: bioinsetticida a base di Beauveria bassiana.- Informatore 

Fitopatologico, 4: 61-64. 

BEZZI A., CADEN S., 1991 – Piante insetticide e pesticide.- Erboristeria domani. Ottobre: 65-79. 

CAPELLA A., GUARNONE A., DOMENICHINI P., 2001 – Azadiractina: caratteristiche, attività biologica e strategie di 

impiego su melo e ortive in coltura protetta.- Notiziario sulla protezione delle piante, 13: 59-63. 

CASIDA J.E., PESSAH I.N., SEIFERT J., WATERHOUSE A.L., 1987 – Ryania insecticide: chemistry, biochemistry and 

toxicology.- In Pesticide science and biotechnology. Proceedings of the 6 th international congress of pesticide 

chemistry., Ottawa, Canada, 10-15 August 1986: 177-182. 

DEL BENE G., GARGANI E., LANDI S., 2000 – Evaluation of plant extracts for insect control.- Journal of Agriculture and 

Environment for International Development, 94(1): 43-61. 

ISMAN M.B., KOUL O., ARASON J.T., STEWART J., SALLOUM G.S., 1991 – Developing a neem-based insecticide for 

Canada.- Memoirs of the Entomological Society of Canada, 159: 39-47. 

LA TORRE A., ALEGI S., IMBROGLINI G., 2002 – Mezzi di difesa in agricoltura biologica.- Informatore Agrario, 16 

Suppl.1: 4-42. 

MAGNANO DI SAN LIO G., VACANTE V., 1989 – I funghi entomopatogeni nella lotta biologica contro i fitofagi.- 

Informatore Fitopatologico, 11: 17-25. 

ROVESTI L., DESEO K.V., 1990 – Azadirachta indica A Juss (Neem) e sue potenzialità nella lotta contro gli insetti.- 


SCHMUTTERER H., 1990 – Properties and potential of natural pesticide from the neem tree, Azadirachta indica.- Ann. 

Revue Entomology: 271-297. 

TREMBLAY E., 1985 – Entomologia Applicata. Vol. 1. Generalità e mezzi di controllo. Liguori Ed., Napoli: 203 pp. 

TSOLAKIS H., LETO G., RAGUSA S., 1997 – Effects of some materials on Tetranichus urticae Koch (Acariformes, 

Tetranichidae) and Typhlodromus exhilaratus Ragusa (Parasitiformes, Phytoseiidae).- 4th International 

Conference on pest in agriculture, 6-8 January, Le Corum, vol. I: 239-245. 


75

12 

DETERMINATION OF Cd (II), Cu (II), Pb (II), AND Zn (II) IN BIOLOGICAL AND NOT BIOLOGICAL 

CITRUS ESSENTIAL OILS BY DERIVATIVE POTENTIOMETRIC STRIPPING ANALYSIS (dPSA) 

Dugo Giacomo, Giuffrida Daniele, Drogo Alessandra, La Pera Lara 

Dipartimento di Chmica Organica e Biologica Università di Messina Salita Sperone, 31, 90166 S. Agata -Messina 

Citrus essential oils are complex mixtures of many classes of volatile (85-98%) and not volatile (15-2%) 

compounds (1); they are used in food and cosmetic farm as aromatizer and in pharmaceutical industry as additives of 

some drugs (2). Their wide use implies a strict control of the presence of organic and inorganic contaminants as heavy 

metals. There is a lack of available data regarding to the presence of heavy metals: some reports concerning to the 

micro-elements composition of citrus peel extracts were found (3,4). Metals levels in citrus essential oils mostly 

depend on the type of soil, agrochemical treatments, but also on extraction procedures as scraping or pressing, since 

the fruits inevitably come in contact with metallic surfaces (5). In this work potentiometric stripping analysis is used to 

determine the content of Cd (II), Cu (II), Pb (II) and Zn (II) in lemon, mandarin, orange and bergamot non biological 

essential oils produced in Sicily and Calabria in the crop year 1999 (6). Moreover 13 samples of bergamot biological 

essential oils and 13 samples of non-biological essential oils produced in Calabria in the crop year 2000, were studied. 


Reagents 

All oils are sampled in dark glassy bottles, with blind nipples, and stored at 4°C until the analyses. Ultra pure 

hydrochloric acid (34-37%), Hg (II) (1000 µgmL -1 , 1M in hydrochloric acid) and Cd (II), Cu (II), Pd (II), Pb (II), Zn (II) 

(1000 µgmL -1 , 0.5 N in HNO3) standard solutions were purchased by Panreac (Barcelona, Spain); Ga (NO3)3⋅3H2O (5 g, 

99.9%) was purchased by Aldrich Chem. Co. (Milwakee, WI, USA). The extracts were filtrated on a carbon column 

Supelclean ENVI-Carb SPE (0.5 g, 6 mL), purchased by Supelco (Bellefonte, PA, USA). 

Apparatus 

Metals analysis are carried out by a PSA ION 3 potentiometric stripping analyzer (Steroglass, S. Martino in Campo, 

Perugia, Italy), connected to an IBM-compatible personal computer. The analyzer operates under the control of the 

NEOTES 2.0.1 software package (Steroglass). The determination is carried out in a conventional three-electrode cell. 

The working electrode was a glassy carbon one coated with a thin mercury film; reference electrode was an Ag/AgCl 

electrode (KCl 3M), and a platinum wire auxiliary electrode was used. 

Procedures 

Hydrochloric acid extraction 

A 8.60 g (10.00 mL) sample aliquot and a 10 mL volume of 36% ultrapure-hydrochloric acid (Panreac, Barcellona), are 

introduced in a teflon beaker. The extraction was carried out for about 30 min under magnetic stirring at the temperature 

of 90 °C. The acid sample, was transferred in a separating funnel and let to cool for about 5 minutes. In order to favour 

the separation of the two phases, the mixture is spiked with 1.0 mL ultrapure methanol, then the acid layer was collected 

in a 20.00 mL flask. The organic layer is extracted again with 4.50 mL of concentrated hydrochloric acid for 5 min, 

under the same conditions described, and then washed with 4.50 mL of boiling water; washings were added to the acid 

extracts and made up to the mark with water. The obtained solution was passed through a carbon column. The filtered 

solution was used for the simultaneous determination of Cd (II), Cu (II), Pb (II), and Zn (II). 

Potentiometric determination 

All analysis were executed in a conventional three electrodes cell; working electrode was a glassy carbon electrode 

coated with a Hg film, the reference one was an Ag/AgCl electrode, the counter was a Pt electrode. The concentrations 

of Cd (II), Cu (II), Pb (II), and Zn (II) were simultaneously determined. 1.00 mL of acid extract, 17.50 mL of ultrapure 

water, 1.00 mL of 1000 µg mL -1 Hg (II) as oxidant agent and 0.50 mL of 10 µg mL -1 Ga (III) - in order to prevent 

Cu-Zn complexes formation on the mercury film (6)- were put into the electrochemical cell. The quantitative analysis 

was executed by the multiple points standard additions method. The potentiometric parameters are reported on table 1. 

Method performances 


76

Recovery and repeatabilty tests were menaged by emploing the above described method to lemon, mandarin, orange 

and bergamot essential oils. Cd (II), Cu (II), Pb (II), and Zn (II) recoveries always spanned from 95.00-100.50% 

evidencing that metals quantification remained unaffected by the clean-up steps. The repeatability was > 95.00% for 

Cd (II), Cu (II), Pb (II), and Zn (II) in all types of oils. Detection limits obtained for the four analytes, ranged from 

0.10 to 0.98 ng g -1 in lemon, mandarin, sweet orange and bergamot essential oils. 

Application 

Figure 1 show the concentration of Cd, Cu, Pb and Zn in lemon, sweet orange, mandarin in Sicilian and Calabrian 

bergamot essential oils. It’s evident that Cd is the metal present in the lowest amount and Zn in the highest, in all types 

of essential oils. Cd concentration spanned from 2 to 23.3 ng g -1 ; Cu from 17.8 to 380.0 ng g -1 ; Pb ranged from 75.9 to 

180.3 ng g -1 and Zn from 804.5 to 1647.5 ng g -1 . 

Moreover, the concentration of Cd, Cu, Pb and Zn was determined in 13 samples of biological bergamot essential oils, 

and 13 non biological ones. Figure 2 evidences that there was any significant difference between biological and non 

biological oils metals level. 

LITERATURE 

1. Di Giacomo A., Mincione B.Gli oli essenziali agrumari in Italia. 1994 Ed. Laruffa, p.196. 

2. Gorinstein S., Martin-Belloso O., Park Y. S., Haruenkit R., Lojek A., Ciz, M., Caspi A., Libman, I., Trakhtenberg 

S.Comparison of some biochemical characteristics of different citrus fruit. Food Chem. 2001, 74, 309-315. 

3. Simpkins W.A., Honway, L., Wu M., Harrison M., Goldberg D. Trace elements in Australian orange juice and 

other products. Food Chem, 2000, 71, 423-433. 

4. Di Giacomo, A. Tecnologia dei prodotti agrumari. Parte II. 1988, duplicated lecture notes, Univrsità di Reggio 

Calabria, 95-120. 

5. La Pera L., Saitta M., Di Bella G., Dugo G.mo. Simultaneous determination of Cd (II), Cu (II) Pb (II) and Zn (II) in 

citrus essential oils by derivative potentiometric stripping analysis. J. Agric. Food Chem. 2003 in press 

6. La Pera L., Lo Curto S., Visco A., Dugo G.mo. Derivative Potentiometric Stripping Analysis (dPSA) used for 

determination of cadmium, copper, lead and zinc in in Sicilian olive oils. J. Agric. Food Chem. 2002, 50, 3090- 

3094. 


77

ng/g 

1600,00 

1400,00 

1200,00 

1000,00 

800,00 

600,00 

400,00 

200,00 

0,00 

Lemon Mandarin Sweet orange Bergamot 

Figure 1.Metals content of lemon, mandarin, sweet orange and bergamot essential oil from 1999. 

350 

300 

250 

200 

ng/g 

150 

100 

50 

0 

142,0 

200,4 

128,6 

Fig 2: mean metals content in biological and non biological bergamot essential oils produced in Calabria in the 

crop year 2000. 

78 

166,1 

343,2 

Cu Pb Zn 


303,6 

Cd 

Cu 

Pb 

Zn 

Non bio 

Bio

13 

IMPIEGO DEL TIMOLO NEL CONTROLLO DELLA VARROOSI DELLE API. 

EFFICACIA, PERSISTENZA E RESIDUI. 

I. Floris 1 , A. Satta 2 , P. Cabras 3 , A. Angioni 3 

1 Dipartimento di Protezione delle Piante - Sezione di Entomologia agraria, Università di Sassari, Via De Nicola, 07100 

Sassari. 

2 Istituto per lo Studio degli Ecosistemi – Sezione Ecologia applicata e Controllo biologico - CNR Sassari, Via De 

Nicola, 07100 Sassari. 

3 Dipartimento di Tossicologia, Università di Cagliari, Via Ospedale 72, 09124 Cagliari. 

SUMMARY 

During the last years, after Varroa destructor resistance have been detected to some important synthetic acaricides, 

many alternative ways of varroa control based on natural products were tested. According to the literature, thymol 

seems to deserve a strong interest for the treatment of varroa infestation. In this paper, the results of an apiary trial 

conducted during the summer of 2001 in a Mediterranean environment (Sardinia, Italy) were reported. The 

effectiveness of thymol, the persistence in two acaricide formulations (gel - Apiguard and vermiculite – Api Life 

VAR®) and the residues in honey and wax were evaluated. Both the thymol formulations, after the treatments, reduced 

significantly the levels o mite infestations of both adult bees and sealed brood (average of about 90%). However, a 

considerable colony-to-colony variability in effectiveness was recorded. A moderate negative effect of the thymol 

treatments on the colony development was also observed. During two weeks of treatments, the bees removed about all 

the applied product (gel or vermiculite). Residues found in honey varied from 0.40 and 8.80 mg/Kg for Apiguard and 

from 0.12 and 4.03 mg/Kg for Api Life VAR®, but according to EU regulation No. 2377/90 thymol is a non-toxic 

veterinary drug, which do not need a MRL (maximal residue limit). The wax residues reached relatives higher levels 

than honey since thymol is a fat-soluble ingredient (average of 21.6±13.0 and 147.7±188.9 for Api Life VAR and 

Apiguard, respectively). However, after the treatment, thymol rapidly evaporates from wax. 

INTRODUZIONE 

L’acaro Varroa destructor Anderson & Trueman, agente della Varroosi delle api, rappresenta il più temibile parassita di 

Apis mellifera L. ed il suo controllo in apiario impone il ricorso a trattamenti acaricidi, spesso mediante l’impiego di 

sostanze di sintesi (Acrinathrine, Clorbenzialate, Chlorfenvinphos, Chlordimerform, Fenotiazine, Bromopropilate , 

Fluvalinate, Amitraz, Coumaphos, Cymiazole, Flumethrin, Tetradifon), con conseguenti problemi di farmaco-resistenza 

e di residui nei prodotti dell’alveare, già segnalati in letteratura (Milani, 1999; Wallner, 1999). 

Negli ultimi anni, si sta affermando, a livello mondiale, l’impiego di sostanze di origine naturale, ed in particolare di 

alcuni acidi organici (acido formico, acido ossalico e acido lattico) e del timolo (Imdorf et al., 1999; Calderone, 1999; 

Whittington et al., 2000). 

Nel presente lavoro, è stata studiata l’efficacia del timolo nel controllo della Varroosi in un ambiente mediterraneo, 

valutandone altresì i residui nel miele e nella cera e verificandone la persistenza nelle due diverse formulazioni 

commerciali impiegate. 

Materiali e Metodi 

Prova in apiario 

La prova è stata condotta in un apiario della Sardegna centro-meridionale (Oristano) nel periodo giugno-luglio del 2001. 

Una postazione di 15 alveari con arnie Dadant-Blatt standard da 10 favi e colonie derivate da Apis mellifera ligustica 

Spin., è stata ripartita in 3 gruppi di 5 alveari ciascuno, omogenei per consistenza ed infestazione, valutate, 

rispettivamente, mediante rilievi preliminare sulla superficie di covata e tramite campionamento di api adulte (300 api 

per alveare) e di covata opercolata (300 cellette per alveare ) (Floris et al., 2001). 

Per il trattamento sono state utilizzate due formulazioni a base di timolo: Api Life VAR® e Apiguard. La prima, 

confezionata in tavolette di vermiculite da circa 12 g contenenti circa il 74% di timolo (pari a circa 9 g di timolo per 

tavoletta), il 3,7% di mentolo, il 3,7% di canfora e il 16% di eucaliptolo; mentre nella seconda formulazione il timolo 

era incluso in gelatina nella percentuale del 25% e confezionato in vaschette contenenti 50 g di gel (12,5 g di timolo). 

Un gruppo di alveari è stato trattato con Api Life VAR, uno con Apiguard ed il terzo è stato tenuto come testimone. Il 

trattamento è stato eseguito il 21 Giugno 2001 impiegando rispettivamente due mezze tavolette di Api Life VAR ed una 

vaschetta di timolo in gel per alveare. Un secondo trattamento è stato effettuato il 6 Luglio 2001. 


79

Durante l’esperimento, per due volte la settimana, è stato eseguito il conteggio delle femmine di Varroa cadute sul 

fondo dell’arnia. Alla fine della prova sono stati nuovamente eseguiti i rilievi, per valutare consistenza e livello di 

infestazione degli alveari trattati e testimone. 

Prima, durante e dopo il trattamento, sono stati inoltre eseguiti i prelievi di miele e di cera da ciascun alveare trattato e 

testimone, nonché dei formulati dai soli alveari trattati, per valutare la persistenza e i residui del timolo. 

I campioni di miele (~ 5 g per campione) sono stati prelevati da 100 cellette disopercolate scelte a caso da tre favi del 

nido di ciascun alveare, aspirandolo con una siringa. Il campione di cera (~ 10 g) è stato ottenuto tagliando 3 porzioni di 

circa 2 cm 2 prelevati da diversi favi del nido di ciascun alveare. Infine, i prelievi delle formulazioni sono stati effettuati 

asportando un frammento di gelatina o di vermiculite. I campioni sono stati conservati in freezer (-18°C) fino al 

momento dell’analisi. 

L’efficacia dei trattamenti è stata valutata impiegando come parametro la percentuale di mortalità M (Henderson e 

Tilton, 1955) calcolata come: 

⎡ ⎛ Bc ⋅ At ⎞⎤ 

M = 100 ⋅ ⎢1 

− ⎜ ⎟⎥ 

⎣ ⎝ Bt ⋅ Ac ⎠⎦ 

dove Bc e Bt rappresentano la percentuale di infestazione (degli adulti o della covata opercolata) prima del trattamento, 

rispettivamente negli alveari testimone (Bc) e in quelli trattati (Bt); Ac e At rappresentano la percentuale di infestazione 

dopo il trattamento, rispettivamente negli alveari testimone e in quelli trattati. 

Reattivi 

Gli standard analitici del Timolo (98%), Cineolo (99%), Mentolo (96%) e Canfora (96%) sono stati ottenuti dalla 

Aldrich Chemical, Janssen (Geel, Belgium), Carlo Erba (Milano Italia), rispettivamente. Le soluzioni madre (1000 

mg/Kg) sono state preparate in acetone (SupraSolv, Merck, Darmstadt, Germany). Le soluzioni di lavoro sono state 

preparate giornalmente, per diluizione con estratti in dietil etere di miele e cera non trattati (controlli). Il dietil etere era 

solvente per analisi (Merck, Darmstadt, Germany). 

Procedure di estrazione 

Formulati - Un g di ciascun formulato è stato pesato in un provettone da 50 ml con tappo a vite, venivano poi aggiunti 

20 ml of metanolo (Chromanorm per HPLC, Prolabo) ed il protettone agitato (15 min) in un agitatore rotante e poi 

sonificato per 5 min in un bagno ad ultrasuoni (Transsonic T460). Un’aliquota di metanolo (100 µl) veniva diluita a 5 

ml con acetone ed iniettata in GC. 

Miele - Un g di miele veniva pesato in un provettone da 15 ml con tappo a vite e veniva solubilizzato con 2 g d’acqua, 

venivano poi aggiunti 2 ml di etere dietilico, ed il protettone agitato (10 min) in un agitatore rotante. Dopo separazione 

delle fasi, un’aliquota dell’estratto era iniettata in GC. 

Cera - Si pesavano 0,5 g di cera in un provettone da 25 ml con tappo a vite, si aggiungevano 5 ml di miscela 

metanolo/acqua (1:1) ed il provettone veniva immerso in un bagnomaria a 70 °C fino a dissoluzione della cera, e quindi 

agitato in vortex per 1 min. Dopo raffreddamento a temperatura ambiente, venivano aggiunti 5 ml di etere dietilico, ed il 

provettone sottoposto ad agitazione per 15 min. in agitatore rotante. Dopo sepa-razione delle fasi, l’estratto etereo 

veniva centrifugato ed un’aliquota iniettata in GC. 

Recuperi 

Campioni non trattati di miele e cera venivano fortificati con 0,1, 0,5, 2,0 e 5,0 mg/Kg of timolo, mentolo, cineolo e 

canfora, ed analizzati secondo le metodiche descritte precedentemente. Le analisi sono state replicate quattro volte per 

ogni livello di fortificazione. La media dei recuperi ottenuti per il miele era 97% (range 84-111%) con un coefficiente di 

variazione massimo (CV) del 5,6%; e per la cera 89% (range 82-98%) con un coefficiente di variazione massimo (CV) 

di 8,5%. 

Gascromatografo 

I campioni sono stati analizzati usando un gascromatografo HRGC 5160 Serie Mega equipaggiato con un detector FID 

80 , un’autocampionatore AS 800, un’iniettore split-splitless (Carlo Erba, Milano, Italia), e collegato ad un integratore 

HP 3396 A (Hewlett Packard, Avondale, PA, USA). La colonna era una capillare in silice fusa DB-5 MS (5% Phenyl- 

Methyl-Polysiloxane) 30 m x 0.25 mm id e 0.1µm di spessore del film (J&W Scientific, Folsom, CA). L’iniettore ed il 

detector erano tenuti ad una temperatura di 100 °C e 200 °C, rispettivamente. Le analisi dei formulati venivano eseguite 

iniettando 2 µl in modalità split, con rapporto di splitaggio di 1:10, mentre tutti gli altri campioni venivano analizzati 

iniettando 2 µl in modalità splitless (30 s). La temperatura del forno era programmata come segue: 60 °C (5 min), salita 

fino a 130 °C (2 °C/min) ed isoterma per 4 min, salita fino a 180 °C (10 °C/min). L’elio era il gas di trasporto e di 

makeup a 1,8 e 30 ml/min, rispettivamente. La fiamma del FID era alimentata da aria ed idrogeno a 250 e 30 ml/min, 

rispettivamente. Le rette di taratura venivano calcolate fra le altezze dei picchi e le concentrazioni utilizzando il metodo 


80

dello standard esterno. E’ stata trovata una buona linearità nel range 0,1-5,0 mg/Kg con un coefficiente di correlazione 

di 0,9994. Il cleanup non era necessario in quanto non vi erano picchi interferenti. Il limite di determinazione (Their and 

Zeumer, 1987) era di 0,1 mg/Kg. 

Analisi Statistica 

I dati acquisiti durante la sperimentazione sono stati sottoposti ad analisi statistica (ANOVA) previa trasformazione 

angolare (arcsin √y/100), nel caso di valori percentuali, al fine di ridurre l’eterogeneità della varianza. Nelle tabelle e 

nelle figure sono riportati I valori non trasformati. Quando I test F risultavano significativi, le medie sono state separate 

applicando il test della Differenza Minima Significativa (LSD con α = 0,05). 

RISULTATI E DISCUSSIONE 

Efficacia dei trattamenti 

La tabella I evidenzia come, prima dell’ inizio degli esperimenti, nessuna differenza statisticamente significativa fosse 

riscontrabile fra i tre gruppi sperimentali, relativamente alla superficie di covata opercolata ed ai livelli di infestazione. 

La tabella II riporta i valori per gli stessi parametri registrati al termine del trattamento. 

Tab. I – Infestazione e consistenza degli alveari prima del trattamento. 

Gruppo di Alveari Infestazione covata Infestazione api adulte Superficie di covata 

opercolata (%) 

(%) 

(cm 2 ) 

Api Life Var® 2,6 ± 1,2 3,1 ± 1,7 2.387 ± 1.071 

Apiguard 2,6 ± 1,0 2,8 ± 1,0 2.425 ± 1.128 

Testimone 2,8 ± 2,2 2,1 ± 0,9 2.368 ± 977 

Le medie nella stessa colonna non sono significativamente differenti (ANOVA, P > 0,05) 

Tab. II – Infestazione e consistenza degli alveari dopo il trattamento. 

Gruppo di Alveari Infestazione covata Infestazione api adulte Superficie di covata 

opercolata (%) 

(%) 

(cm 2 ) 

Api Life Var® 0,7 ± 0,5 a 0,4 ± 0,5 a 902 ± 319 a 

Apiguard 1,7 ± 0,6 a 1,5 ± 1,0 a 1.090 ± 263 a 

Testimone 8,4 ± 4,8 b 8,0 ± 6,1 b 1.598 ± 282 b 

Le medie nella stessa colonna seguite dalla stessa lettera non sono significativamente differenti (ANOVA, P > 0,05 LSD 

test) 

I livelli di infestazione, sia della covata sia degli adulti, sono diminuiti nei trattati ed aumentati nel testimone, mentre la 

superficie di covata opercolata è diminuita in tutti e tre i gruppi anche se in misura maggiore nei trattati. Le differenze 

riscontrate sono risultate statisticamente significative solo tra i trattati ed il testimone. Tali differenze, evidenziano un 

effetto del timolo sulla consistenza delle colonie, con una flessione più marcata negli alveari trattati rispetto ai 

testimoni. 

L’efficacia, espressa in termini di percentuale di mortalità, nel gruppo trattato con Apiguard, è risultata pari a 90,4 ± 

8,3% ed a 95,5 ± 8,7%, considerando, rispettivamente, il livello di infestazione della covata e degli adulti. Nel gruppo 

trattato con Api Life VAR®, considerando i medesimi parametri nello stesso ordine, sono stati riscontrati valori pari a 

74,8 ± 13,1% e di 81,3 ± 15,5%. Tali differenze non sono risultate statisticamente significative per effetto dell’elevata 

variabilità rilevata all’interno di ogni gruppo, che evidenzia un’azione di tale composto volatile presumibilmente 

condizionata da fattori biologici e microclimatici interni all’alveare. 

L’andamento della caduta degli acari durante l’esperimento, è riportato nella Figura 1. La mortalità più elevata é stata 

riscontrata nel gruppo trattato con Apiguard durante la prima settimana di trattamento e, in seguito alla sostituzione 

delle vaschette, nella terza settimana. 


81

N° di varroe 

700 

600 

500 

400 

300 

200 

100 

0 

17-21/VI 

21-25/VI 

25-28/VI 

Fig. 1- Andamento della caduta degli acari 

Persistenza e residui del timolo 

Apiguard Api Life Var Controllo 

28/VI-2/VII 

2-5/VII 

a) Metodo analitico 

La metodica messa a punto nel presente lavoro per l’estrazione del timolo è risultata estremamente rapida e non ha 

richiesto alcun clean-up rispetto ai metodi riportati in precedenti lavori (Bogdanov et al. 1998, Martel e Zeggane, 2002; 

Nozal et al. 2002), in quanto gli estratti non hanno evidenziato picchi interferenti. The average recovery for honey was 

97% (range 84-111%) with a maximum coefficient of variation (CV) of 5.6%; and for wax it was 89% (range 82-98%) 

with a maximum coefficient of variation (CV) of 8.5%. A good linearity was achieved in the 0.1-5.0 mg/Kg range with 

a correlation coefficient of 0.9994. Cleanup was unnecessary because there were no interference peaks. The limit of 

determination (Their and Zeumer, 1987) was 0,1 mg/Kg. 

b) Residui 

Inizialmente si è proceduto ad un controllo del titolo del timolo in entrambi i formulati. Secondo quanto riportato in 

etichetta una tavoletta di Api Life Var® dovrebbe contenere il 2,52% di vermiculite ed il resto di oli essenziali; un 

controllo da noi effettuato ha accertato la presenza di vermiculite in quantità superiore all’11% (11,4±0,64). A tale 

risultato si è pervenuti pesando dopo essiccamento in stufa, quantità note di Api Life Var®, precedentemente sottoposte 

all’estrazione degli oli essenziali secondo la metodica descritta. I titoli in timolo dell’Api Life Var e dell’Apiguard, 

determinati gas cromatograficamente, sono risultati essere del 56% contro il 74% e del 18% contro il 25% dichiarati. 

I campioni di Api Life Var ed Apiguard, prelevati durante l’esperimento, si presentavano “Propolizzati” (disgregati 

dall’azione delle api). 

I residui di timolo nel miele prelevato dalle arnie trattate con Api Life Var® variano notevolmente da alveare ad alveare 

(Tab. III). Alla fine della prima settimana dal trattamento, la media dei residui di timolo nel miele, prelevato dalle 5 

arnie prese in considerazione, è stata di 1,97 ± 1,54 mg/Kg; tale valore è diminuito nel corso della seconda settimana 

fino a 0,75 ± 0,44 mg/Kg. A questo punto, sostituite le vecchie tavolette, i residui aumentano nuovamente nel corso 

della terza settimana (1,05 ± 1,01 mg/Kg) per decadere nel corso dei sette giorni successivi fino al valore di 0,62 ± 0,57 

mg/Kg. Da tale andamento si evince che il rilascio più abbondante del timolo nelle nuove tavolette viene in parte 

trattenuto dal miele, dal quale tuttavia evapora in parte nell’arco della seconda settimana. 

Nei campioni di miele prelevati dagli alveari trattati con Apiguard, possiamo notare la stessa grande variabilità di 

concentrazione del residuo di timolo, con un’analoga decadenza della concentrazione tra prima e seconda e tra terza e 

quarta settimana di trattamento (Tab. IV). I valori alla fine della prima e della terza settimana sono significativamente 

più elevati rispetto a quelli riscontrati con l’uso di Api Life Var®. Ciò può essere messo in correlazione con la maggior 

concentrazione del timolo nell’Apiguard rispetto all’ Api Life Var® e con le differenti modalità di erogazione di tale 

composto. 

Nel miele prelevato dagli alveari testimone, la concentrazione di timolo, quando presente, è molto modesta (0,11 ± 0,21 

mg/Kg) (Tab. III). 


82 

5-8/VII 

9-12/VII 

12-16/VII 

16-19/VII

Tab. III - Residui di timolo(mg/Kg) nel miele (Media±Dev.St.). 

Formulazione Prelievo a 7gg Prelievo a 14gg Prelievo a 7gg Prelievo a 14gg 

dal 1° tratt. dal 1° tratt. dal 2° tratt. dal 2° tratt. 

Api Life Var® 1,97±1,54 0,75±0,44 1,05±1,01 0,62±0,57 

Apiguard 3,07±1,80 0,89±0,70 2,55±3,50 0,96±0,61 

In generale, i residui nel miele si presentano altamente variabili come già riscontrato da Bogdanov et al. (1998). Tenuto 

conto che si tratta di un composto molto volatile, la variabilità potrebbe essere in funzione delle differenti condizioni 

microclimatiche che possono verificarsi negli alveari in rapporto alle diverse condizioni biologiche delle colonie. 

I residui di timolo nei campioni di cera, prelevati prima e al termine della quarta settimana di trattamento, sono riportati 

nelle tabella IV. 

Tab.IV - Residui di timolo (mg/Kg) nella cera (Media±Dev.St.). 

Formulazione Prima del trattamento Alla fine del trattamento 

Api Life Var® n.d. 21,6±13,0 

Apiguard 1,0±0,7 147,7±188,9 

Nei campioni di cera prelevati prima del trattamento dagli alveari testimone, la concentrazione del timolo, quando 

presente, ha valori pari a 2,9 ± 4,6 mg/Kg, alla fine del trattamento i valori sono di 1,5 ± 0,8 mg/Kg. La medesima 

natura apolare di timolo e cera comporta evidentemente una maggiore presenza del composto nella cera stessa piuttosto 

che nel miele. 

In definitiva, sulla base dei risultati acquisiti nel presente studio, si conferma la buona efficacia del timolo, in 

riferimento alle due differenti formulazioni testate, nel controllo della Varroosi in apiario, con una riduzione percentuale 

media dell’infestazione del 90 % e del 95% circa se riferita alla covata o alle api adulte. Questi risultati, comparati con 

quelli ottenuti con altri acaricidi nello stesso ambiente (Floris et al., 2001; Floris et al., 2001), dimostrano la validità del 

timolo, seppure con una modesta incidenza negativa del trattamento sullo sviluppo delle colonie, come mezzo 

alternativo agli acaricidi di sintesi per il controllo della Varroa, con il vantaggio che, trattandosi di un composto di 

origine naturale, non presenta alcun limite massimo di residuo nel miele (Imdorf et al., 1999; Wallner, 1999). 

BIBLIOGRAFIA 

Bogdanov S., Imdorf A., Kilchenmann V., 1998 - Residues in wax and honey after Apilife Var treatment. Apidologie 

1998, 29, 513-524. 

Calderone N.W., 1999 – Evaluation of formic acid and Thymol-based blend of natural products for fall control of 

Varroa jacobsoni (Acari: Varroidae) in colonies of Apis mellifera (Hymenoptera: Apidae). J. Econ. Entomol., 

92(2): 253-260. 

Floris I., Cabras P., Garau V.L., Minelli E. V., Satta A., Troullier J., 2001 - Persistence and Effectiveness of Pyretroids 

in Plastic Strips Against Varroa Jacobsoni (Acari: Varroidae) and Mite Resistance in a Mediterranean Area. J. 

Econ. Entomol. 2001, 94(4): 806-810. 

Floris I., Satta A., Garau V.L., Melis M., Cabras P., Aloul N., 2001 - Effectivness, persistence, and residue of amitraz 

plastic strips in the apiary control of Varroa destructor. Apidologie 2001, 32, 557-585. 

Henderson C. F., Tilton E. W., 1955 - Tests with acaricides against brown wheat mite. J. Econ. Entomol., 1995, 48, 

157-161. 

Imdorf A., Bogdanov S., Ibáñez Ochoa R., Calderone N.W., 2001 – Use of essential oils for the control of Varroa 

jacobsoni Oud. In honey bee colonies. Apidologie, 30 (2-3): 209-228. 

Martel A.C., Zeggane S. – Determination of acaricides in honey by high-performance liquid chromatography with 

photodiode array detection. Journal of Chromatography A. 954(1-2):173-180, 2002 Apr 19. 

Milani N., 1999 – The resistance of Varroa jacobsoni Oud. to acaricides. Apidologie, 30 (2-3): 229-234. 

Nozal M.J., Bernal J.L., Jimenez J.J., Gonzales M.J. Higes M., 2002 – Extraction of thymol, eucalyptol, menthol, and 

camphor residues from honey and beeswax – Determination by gas chromatography with flame ionization 

detection. Journal of Chromatography A. 954(1-2):207-215, 2002 Apr 19. 

Their H.P., Zeumer H., - 1987 – Manual of Pesticides Residues Analysis: VCH: Weinheim, Germany, Vol.1, pp. 37-74. 

Wallner K., 1999 - Varroacides and their residues in bee products. Apidology, 1999, 30, 235 -248. 

Whittington R., Winston M.L., Melathopoulos A.P., Higo H.A., 2000 – Evaluation of the botanical oils neem, thymol, 

and canola sprayed to control Varroa jacobsoni Oud. (Acari: Varroidae) and Acarapis woodi (Acari: 

Tarsonemidae) in colonies of honey bees (Apis mellifera L., Hymenoptera: Apidae). American Bee Journal, 140 

(7): 567-572. 


83

14 

ASSESMENT OF PHYTOTOXICITY TO GRAPEVINE OF TRADITIONAL AND NEW COPPER 

COMPOUNDS USED IN COPPER REDUCTION STRATEGIES IN ORGANIC VITICULTURE AND ITS 

RELATIONSHIP WITH ENVIRONMENTAL CONDITIONS AND NUMBER OF TREATMENTS 

Ilaria Pertot, Marco Delaiti, Diego Forti 

Istituto Agrario di S. Michele all’Adige, via Mach 1, S. Michele all’Adige, (TN), Italy 

ilaria.pertot@ismaa.it 

Introduction 

In organic viticulture Plasmopara viticola control is based almost exclusively on copper. The expected restrictions on 

copper used in organic agriculture in the European Union have stimulated the research of alternatives and the 

optimisation of copper allowed quantity. To compare efficacy against downy mildew of different copper compounds 

several researches have been done, but phythotoxicity has received scant attention, particularly in relation to copper 

reduction strategies in organic viticulture. Phytotoxicity risk must be taken into consideration during grapevine 

phenological cycle, because copper compounds could affect the quality of grape and the vegetative balance of the plant. 

Researches have shown that, using the same Cu2+ dosage, efficacy and rainfastness of different copper compounds and 

formulations are the same (Pertot et al., 2002). Reduction in annual copper quantity will be possible with a rationale 

use of dosages, that can vary between 30 to 70 g Cu2+/hl during the vegetative stage of the plant, depending on weather 

conditions and infection risk evaluation. The rationale dosages used must be associated with a careful protection against 

primary infections, the execution of treatments before forecasted rain and the increasing of frequency application in the 

period of maximum plant growth. 

Among new copper formulations, compounds and adjuvants only copper peptidate could allow a consistent reduction in 

copper annual quantity, giving at the same time a adequate efficacy level against the disease (Pertot et al., l.c.). 

The aim of this research was: i) the evaluation, with low dosages, of phytotoxicity to grapevine in field of new copper 

formulations compared to the traditional ones; ii) the identification in controlled conditions of the factors that induce 

phytotoxicity. 

Materials and metods 

The trials were done in the experimental organic vineyard of Istituto Agrario di S. Michele all’Adige, located in 

Rovereto (TN, Italy). Chardonnay was the used cv. Test products, reference products and untreated control were 

arranged in a suitable statistical design (fully randomised design, with treatments replicated four times). Products were 

applied using a normal volume of 12 hl/ha, with a four times concentrated sprayer (tab.1). Phytotoxicity assessment was 

done following EPPO standars, guideline PP1/135(2). For each repetition twenty five leaves were randomly collected 

and classified using a scale depending on the symptoms and the percentage of leaf surface affected. 

2001 

2002 

active ingredient commercial name Cu2+ g/ha active ingredient commercial name Cu2+ g/ha 

bordeaux mixture P. Bordolese Dispers 

copper hydroxide Kocide 2000 

copper sulphate Cutril 

cuprous oxide Cobre Nordox 

360, 600, 840 

bordeaux mixture 

copper hydroxide 

copper peptidate 


P. Bordolese Dispers 

Heliocuivre 

Peptiram 7 (2 times) 

Peptiram 7 (3 times) 

360, 600 

150 



Peptiram 5 

Peptiram 7 

300 

150 

cuprous oxide Cobre Nordox 

copper oxychloride ICC0087 

360, 600 

Tab.1 Field treatments in 2001 and 2002 

In greenhouse controlled conditions the influence of temperature (5, 15, 25, 35° C), leaf wetness and number of 

treatments (1, 3, 5, 7) on phytotoxicity of copper peptidate (Peptiram 5) was evaluated. Potted plants, cv. Pinot gris, 

were used. For each treatment were used five plants with two shoots each. Phytotoxicity evaluation was done using 

three classes of intensity: low (from 0 to 0,5 % of leaf surface damaged), middle (from 0,6 to 5%) and high (from 5,1 to 

10 %) and indicated as phytotoxicity index. 


84

phytotoxicity index (%) 

Results 

In field trials, phytotoxicity (fig. 3) was related to particular environmental conditions (which were identified both in 

2001 and 2002 in prolonged leaf wetness), but the degree of damage depended on the copper compound used. Copper 

peptidates, followed by cuprous oxide, induced the highest phytotoxicity level (fig.1). 

Fig. 1 field results in 2001 (left) and 2002 (right). Same letter means that results are not significantly different 

(P=0.05). 


2.5 

2 

1.5 

1 

0.5 

0 



copper sulphate 

cuprous oxide 



In controlled conditions, low temperature, but also high ones, if associated with leaf wetness, induced phytotoxic effects 

(fig. 4), which were evident at cytological level before inducing macroscopic alterations on leaves. At cellular level 

copper precipitate was visible. Chemical composition of precipitates was determined by X ray microanalysis. 

Phytotoxicity increased with the increasing of the number of treatments. 

100 

80 

60 

40 

20 

0 


Fig. 2 Phytotoxicity index (percentage of leaf infected) on plants treated with an increasing number of application at 

different temperature. Effect on dry plants (left) and on wet plants (rightside). 

85 

2.5 

2 

1.5 

a 

0.5 

a a 

a 

5°C 15°C 25°C 35°C 

d 

temperature 

c 

b 

1 

0 


100 

80 

60 

40 

20 

0 


bc 

cd 



d 

cuprous oxide 

b 

copper oxychloride 

5°C 15°C 25°C 35°C 

temperature 



a 

0 

1 

3 

5 

7

Fig 3 – 4 Symptoms of phytotoxicity in field and in greenhouse. 

Discussion and conclusion 

When long leaf wetness periods combined with low or very high temperatures are frequent it is necessary to be very 

careful in using copper compounds, as copper peptidate. Copper peptidate, which could be useful to reduce the annual 

allowed copper amount per hectar, must be used only when grapevine is less sensible to copper phytotoxicity, avoiding 

application until the end of flowering. Copper peptidate repeated treatments whithout periods of rains increase the risk 

of phytotoxicity. In this case could be useful to reduce dosages in the following treatments. 


This work was funded by the Italian Ministry of Agriculture (MiPAF), research project “La difesa delle colture in 

agricoltura biologica”. 

References 

I. Pertot, M. Delaiti, E. Mescalchin, M. Zini, D. Forti (2002) Attività antiperonosporica di nuove formulazioni di 

composti rameici utilizzati a dosi ridotte e prodotti alternativi al rame impiegabili in viticoltura biologica, Atti giornate 

fitopatologiche 2002. 

AA.VV. (1997) Guidelines for the efficacy evaluation of plant protection products, vol. 1-2 OEPP/EPPO, France. 


86

15 

ORGANIC WHEAT QUALITY AND PRODUCTION: THE RESULTS OF THREE YEARS OF TRIALS 

Fabio Fusari, Antonella Petrini 

The relevant increasing of farms and areas interested in organic productions in Italy draws attention to the need of an 

experimentation which can help this sector for an equilibrate growth. CERMIS (Research and Experimental Centre for 

Plant Improvement “N. Strampelli”), in collaboration with ASSAM (Agency for Food and Agriculture Services in 

Marche region), has begun in 1998 an experimental project in the sector of autumn-vernal cereals in which the main 

objectives are identifying the varieties to employ and optimizing the cultivation techniques. A correct varietal choice is 

essential, in organic agriculture, in order to obtain acceptable productive and qualitative results. The morphological and 

physiological characteristics which make cultivars suitable for this kind of cultivation are: rusticity, resistance or 

tolerance to the main phytopathies, ability of competition with weeds and greater efficiency in the use of nitrogen. An 

other aspect to consider, especially in the case of bread wheat, is the qualitative one, because the milling industry 

requires distinct technological characteristics of the stocks according to the different kinds of product (bread, biscuits, 

‘panettone’, etc.). Qualitative characteristics, intrinsic in a specific variety, are also affected by pedological, climatic 

and agronomic factors; so the evaluation of each single variety within a cultivation system is fundamental, especially 

when the external inputs are reduced as in the case of organic agriculture. 

The activity within the project 

Since from 1998 a parcel trial for the varietal comparison of bread wheat has been carried out by an organic farm 

situated along the valley of Potenza river (Macerata, Italy) on a medially fertile, tendentially clayey, soil. The 

cultivation techniques which have been adopted are the ones usually employed by that farm, admitted by the EC Reg. 

2092/91. The main agronomic and qualitative attributes have been taken and statistically analysed in accordance with 

the methodologies adopted by the wheat national network in Italy. The twenty bread wheat varieties that have been 

evaluated have been chosen, between the ones registered to the national catalogue, on the basis of their agronomic, 

merchandise (qualitative classes) and commercial characteristics. Also the old cultivar Abbondanza has been inserted in 

the trial, in order to verify its behaviour in the case of an organic method of cultivation. 

Table 1 - Production indexes (*) of the tested varieties 

Variety name 1999 2000 2001 average 

ABBONDANZA 108 101 134 114 

EUREKA 101 109 135 115 

SERIO 113 104 108 108 

CENTAURO 109 111 97 105 

BOLERO 93 110 94 99 

COLFIORITO 107 92 90 96 

SAGITTARIO 86 99 101 95 

ENESCO 91 101 87 93 

MIETI 109 95 63 89 

PANDAS 103 113 36 84 

SIBILLA 87 98 73 86 

VALLEROSA 95 86 67 83 

SANGIACOMO 98 81 61 80 

ETECHO 157 157 

GUADALUPE 140 140 

TREMIE 128 128 

TIBET 124 124 

GENIO 118 118 

BILANCIA 109 109 

SIRMIONE 78 78 

Field average (t/ha) 3,39 2,75 2,11 2,65 

(*) 100= field average 

YEAR 

Results 

From the analysis of mean results of the thirteen varieties common to the three years of trials, it’s evident that a 

progressive loss of yields over the years (graph 1), due mainly to the anomalous meteorological trend during the two 

last grain seasons, has taken place. 

87 

4,0 

3,5 

3,0 

2,5 

2,0 

1,5 

1,0 

0,5 

0,0 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 

Graph 1 - Average yield (t/ha) of the 13 varieties 

common to the three years of trials 

1999 2000 2001 

Graph 2 - Mean alveographic parameters (W 

and P/L) during the three years of trials 

w P/L 

1999 2000 2001 


1,0 

0,9 

0,8 

0,7 

0,6 

0,5 

0,4 

0,3 

0,2 

0,1 

0,0

In particular the farming year 2000/2001 has been characterized by a mild and humid winter and by low temperatures in 

April, which have damaged the early varieties and the cultivars that are susceptible to some fungal diseases, mainly 

yellow and brown rust. 

In the table 1 the production indexes of the varieties tried out over the three-year period 1998-2001 are summarized. 

The cultivars which highlight the best performances are Abbondanza, Eureka and Serio which, in that period, have 

always given indexes above average. Also the mean index of the variety Centauro is above 100, while the production 

index of the cultivar Pandas decreases dramatically because of the chilling damages suffered during the grain season 

2000/2001. 

Among the varieties tested only during the farming year 2000/2001 the index of the cultivar Sirmione is below average, 

while Etecho and Guadalupe are in evidence for their very high indexes, even if for these varieties it is necessary to wait 

for further experimental evidences. 

Qualitative characteristics 

On a grain sample, mixture of the three repetitions, technological analyses (alveogram, falling number, protein and 

gluten content) have been performed in order to establish the qualitative characteristics of the harvested product. 

Examining the graph 2, where the mean data of the thirteen varieties common to the three years of trials are reported, it 

can be observed that W values are basically low, whereas the ratio P/L indicates a tendential imbalance because of an 

excess of tenacity. Probably this qualitative decay is due to an unfavourable seasonal trend both in 1998/1999 and in 

2000/2001, and to a reduced nitrogen availability during the main stages of the vegetative cycle. Only in the grain 

season 1999/2000 the quality appears medially sufficient, so that in this case most of the varieties can be set in the 

respective qualitative classes according to the Synthetic Quality Index (ISQ) calculated on the basis of the protein 

content and of the alveographic and farinographic characteristics. 

Conclusions 

In general the results which have been obtained demonstrate that organic agriculture gives the chance for satisfying 

yields combined to a middle-low technological quality, anyhow sufficient for the production of biscuits and 

breadmaking flours. The improvement of technological quality for the wheat cultivated with the organic methods 

requires a careful varietal choice, and the use of techniques able to guarantee an adequate availability of assimilable 

nitrogen in the soil. 

*CERMIS – Centro Ricerche e Sperimentazione per il Miglioramento Vegetale “N. Strampelli”, Tolentino (MC) Email: 

cermis@tin.it 


88

16 

IL CONTROLLO DELLA “ MOSCA DELLE OLIVE” (BACTROCERA OLEAE Gmel.) CON METODI 

CONSENTITI IN COLTIVAZIONE BIOLOGICA 

N. Iannotta 

Istituto Sperimentale per l’Olivicoltura, Rende (CS) 

E-mail: n.iannotta@irsaolivicoltura.it 

Introduzione 

Una cultura ecologista ed igienico-salutista recentemente instauratasi in Italia ed in altri Paesi europei e lo stimolo degli 

incentivi comunitari, ha portato un notevole flusso di olivicoltori in direzione della coltivazione biologica. Le superfici 

olivicole investite in questa forma di coltivazione si sono enormemente dilatate in questi ultimi anni, per effetto della 

accresciuta domanda di prodotti “certificati” e per l’aumentato interesse economico verso un tipo di produzione in 

grado di avvantaggiarsi dell’ulteriore valore aggiunto del “biologico”. Tale incremento di superfici investite nel 

comparto olivicolo, già di per sé economicamente importante in Italia (soprattutto nel Mezzogiorno), ha interessato 

nell’ultimo decennio diverse decine di migliaia di ettari e recenti indagini riferiscono che ben il 10% dell’olivicoltura 

convenzionale ha convertito la propria produzione ai sistemi biologici. 

La difesa fitosanitaria in coltura biologica, specialmente negli ambienti meridionali, si pone come un oggettivo limite 

alla competitività economica dell’olivicoltura, soprattutto in considerazione della lotta alla “mosca delle olive”, il più 

dannoso parassita dell’olivo, della quale non si può prescindere. 

Come è ben noto, i prodotti fitosanitari impiegabili in biologico sono contenuti nell’allegato II B del Reg. CEE 2092/91, 

ma le notizie reperibili in letteratura circa la loro efficacia, il loro impatto ambientale ed il loro impatto tossicologico 

sulla qualità del prodotto sono alquanto scarse ed incomplete. 

Da diversi anni l’Istituto Sperimentale per l’Olivicoltura ha avviato specifiche ricerche tendenti a verificare la reale 

efficacia di alcuni prodotti compresi nella citata tabella, ed in qualche caso anche la presenza di residui sul prodotto. In 

questa sede si illustrano le prove effettuate nell’ultimo biennio, concernenti il trappolaggio massale e la lotta al dittero 

con principi attivi ammessi in coltivazione biologica. 

Materiali e metodo 

Le prove di mass-trapping si sono svolte negli anni 2000-2001 nel campo sperimentale dell’Istituto Sperimentale per 

l’Olivicoltura in Rende (CS), dove la “mosca” è presente in maniera massiccia causando notevoli danni, costituito da 

piante giovani (10-15 anni) di cultivar diverse. In questa ricerca è stato sperimentato il dispositivo bersaglio “Attract 

and Kill” (Suneco s.r.l). Esso è stato applicato manualmente alle piante di olivo, in numero di 100 x Ha (33 con 

bicarbonato d’ammonio e feromone; 67 con solo bicarbonato d’ammonio), in luglio dei due anni, e mantenuto fino alla 

raccolta (fine ottobre). Il campo così predisposto (tesi A) è stato confrontato con un’altra parcella (tesi B) trattata con 

metodo antidacico convenzionale (dimetoato al superamento della soglia di intervento), e con una parcella testimone 

(tesi T), trattata solo con acqua. Nella parcella trattata convenzionalmente sono stati effettuati due trattamenti, uno ai 

primi di settembre e l’altro ai primi giorni di ottobre, con dimetoato alla concentrazione di 60gr/hl di p.a. 

In un’altra parcella della stessa azienda è stato effettuato un trattamento con rame (ossicloruro di rame allo 0,5%). 

Le prove di lotta con rotenone e azadiractina sono state effettuate negli anni 2000-2001 nel campo sperimentale 

dell’A.R.S.S.A. in Mirto-Crosia (CS). Ai fini della difesa fitosanitaria, il campo è da diversi anni trattato con metodi 

compatibili con la coltivazione biologica. Esso è stato suddiviso in tre parcelle corrispondenti ad altrettante tesi: A, 

trattata con rotenone (p.c. ROTENA) alla dose di 300 cc/hl, addizionato da 300 g/hl di olio bi anco e 50g/hl di bagnante; 

B, trattata con azadiractina A (p.c. DIRACTIN) alla dose di 150 cc/hl, addizionata con 50g/hl di bagnante; C, trattata 

con sola acqua. I trattamenti sono stati effettuati, alle prime ore del mattino, al superamento della soglia del 20% di 

infestazione attiva. Essi sono stati effettuati il 25 settembre del 2000, unico trattamento dell’anno, ed il 7 settembre e 4 

ottobre del 2001 (due trattamenti). 

I rilievi, per tutte le tesi, sono stati effettuati a scadenza decadica (da luglio a fine ottobre), ed hanno riguardato: a) 

andamento della popolazione adulta presente in campo, per ogni tesi, rilevato mediante monitoraggio con cartelle 

cromotropiche; b) andamento dell’infestazione attiva, per ogni tesi, rilevato mediante campionamento a random ed 

esame di 100 drupe. 


89

Risultati 

Nelle figg.1-2 sono riportati i dati (media nel biennio) riferiti all’andamento dei voli e dell’infestazione attiva nelle 

diverse tesi della prova di mass-trapping. 

Dalla fig. 1 si desume che la maggior presenza del fitofago si registra nella tesi testimone, dove ai rilievi del 15 e del 23 

ottobre raggiunge rispettivamente il numero di 81 e 192 esemplari catturati in media per trappola. Nello stesso periodo 

più contenute appaiono le catture delle rimanenti tesi, con valori fra loro non significativamente differenti. La fig. 2 

mostra i risultati relativi all’infestazione attiva rilevata nelle diverse tesi fino al 25 ottobre, epoca ritenuta ottimale per la 

raccolta nell’azienda dove si è svolta la prova. I dati relativi alle tesi trattate (A e B) evidenziano come l’andamento 

dell’infestazione attiva, da settembre in poi, sia in queste parcelle inferiore rispetto al testimone. Nel confronto fra esse 

si nota un migliore andamento della tesi A fino alla prima decade di ottobre; successivamente, a seguito del trattamento, 

la curva della tesi B subisce una flessione riportando l’andamento dell’infestazione attiva inferiormente a quella della 

tesi A. 

Le figg. 3-4 mostrano i dati relativi al trattamento con il rame (tesi A), posti in confronto al trattamento convenzionale 

con dimetoato (tesi B) e con il testimone (tesi T). 

n°catture x trappola 

200 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 

2/7 

12/7 

22/7 

2/8 

13/8 

23/8 

3/9 

13/9 

La fig.3 evidenzia una efficacia dei trattamenti contro gli adulti, con effetti simili tra loro (rame e dimetoato), come 

dimostrano gli andamenti delle catture inferiori nelle tesi trattate rispetto al testimone. Dall’esame della fig. 4 si desume 

che la tesi trattata con ossicloruro di rame mostra un andamento inferiore al testimone e, per oltre un mese, anche 

rispetto alla tesi trattata con dimetoato. 

Nella fig.5 si rappresentano gli andamenti della popolazione adulta nel campo della prova con 

impiego di rotenone e azadiractina. 

Catture 

n° catture 

200 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 

120 

100 

80 

60 

40 

20 

0 

Tesi A 

Tesi B 

Tesi T 

Fig.5: Andamento della popolazione adulta negli anni in osservazione. 

24/9 

4/10 

15/10 

Fig.1: Andamento della popolazione adulta (catture) nelle diverse 

tesi. 

Tesi A 

Tesi B 

Tesi T 

Trattamento 

25/10 

2/7 12/7 22/7 2/8 13/8 23/8 3/9 13/9 24/9 4/10 15/10 25/10 

Fig.3: Andamento della popolazione adulta (catture) nelle diverse tesi 

Anno 2000 

trattamento 

8/7 18/7 7/8 17/8 28/8 7/9 18/9 28/9 9/10 20/10 30/10 9/11 20/11 30/11 

Tesi A (Rotenone) Tesi B (Azadiractina) Tesi C (Controllo) 

90 

% 

Catture 

80 

70 

60 

50 

40 

30 

20 

10 

25 

20 

15 

10 

0 

5 

0 

20 

15 

10 

5 

0 

3/7 

2/8 

Tesi A 

Tesi B 

Tesi T 

13/8 

23/8 

3/9 

13/9 

24/9 

4/10 

15/10 

Fig.2: Andamento dell'infestazione attiva (uova, larve,pupe) nelle 

diverse tesi. 

10/7 

17/7 

24/7 

Tesi A 

Trattamento 

Tesi B 

2/8 9/8 16/8 23/8 30/8 6/9 13/9 20/9 27/9 4/10 11/10 18/10 25/10 

Fig.4. Andamento dell'infestazione attiva 

(uova, larve, pupe) nelle diverse tesi. 

31/7 

7/8 

14/8 

Anno2001 

trattamento 

21/8 

28/8 

Tesi A (Rotenone) Tesi B (Azadiractina) Tesi C (Controllo) 

4/9 

11/9 

18/9 

25/9 

2/10 

25/10 

trattamento 


Tesi T 

9/10 

16/10 

23/10

Nel primo anno si può notare un andamento caratterizzato da picchi di presenza di adulti notevoli, registrati nel mese di 

agosto, probabilmente a causa delle favorevoli condizioni ambientali, ma dopo il trattamento è visibile l’effetto del 

rotenone che ha ridotto la presenza in campo del dittero sia rispetto alla tesi controllo che rispetto alla tesi trattata con 

azadiractina. 

Nel secondo anno, con le diverse condizioni climatiche, i picchi di presenza si registrano in settembre ed ottobre, 

epoche in cui sono stati effettuati i trattamenti. Questi ultimi hanno prodotto, per entrambi i prodotti saggiati, una 

notevole riduzione delle catture nelle parcelle trattate. 

La fig.6 mostra i risultati ottenuti nei due anni nelle diverse tesi, in relazione all’infestazione attiva. L’esame 

complessivo dei dati indica l’efficacia dei trattamenti, sia relativamente al rotenone che all’azadiractina, nel 

contenimento delle infestazioni daciche. Infatti, in entrambi gli anni in osservazione, i trattamenti hanno prodotto 

sempre una riduzione dell’infestazione attiva contenendone i valori intorno al 20%, fino al momento della raccolta. 

Nella tesi controllo, nelle stesse circostanze, i valori registrati sono risultati più alti, ben oltre il limite del 20%. 

% 

100 

80 

60 

40 

20 

0 

21/8 

11/8 

Carolea (2000) 

Trattamento 

31/8 

20/9 

10/9 

10/10 

30/9 

Fig. 6: Andamento dell’infestazione attiva nel biennio 2000-2001 

20/10 

30/10 

Tesi A (Rotenone) 

Tesi B (Azadiractina) 

Tesi C (Controllo) 

19/11 

9/11 

29/11 

La tab.1 riporta i risultati delle analisi relativamente ai residui di rotenone riscontrati su olive e olio (comunicazione 

personale del professor P. Cabras - Dipartimento di tossicologia dell’Università di Cagliari). 

Tab.1: Residui di rotenone (mg/Kg±SD) riscontrati in olive e olio a diversi giorni dal trattamento. 

Giorni dopo il trattamento Olive Olio 

0 0.99 ± 0.04 — 

2 0.52 ± 0.13 1.89 ± 0.18 

5 0.44 ± 012 1.05 ± 0.12 

9 0.19 ± 0.04 0.51 ± 0.05 

12 0.11 ± 0.02 0.53 ± 0.18 

Ricordando che il limite legale ammesso dalle normative vigenti è di 0.04 mg/Kg di residuo su olive a 10 giorni dal 

trattamento, risulta evidente come tale limite sia stato sempre superato, anche dopo 12 giorni dal trattamento. Inoltre i 

valori ottenuti su olio, ancorché privi di riferimenti legislativi, appaiono particolarmente elevati, indice di una tendenza 

alla liposolubilità del rotenone. 

Conclusioni 

Relativamente al mass-trapping, dai risultati ottenuti nei rilievi effettuati (catture, infestazione attiva), appare evidente 

l’azione di contenimento esercitato dal dispositivo bersaglio “Attract and Kill” contro la “mosca delle olive”. Inoltre, 

ove si considerino anche i benefici effetti conseguibili con questo innovativo metodo (assenza di rischi tossicologici per 

il prodotto, alta selettività di azione contro l’insetto e perciò con bassissimo impatto ambientale, economicità del 

metodo per il ridotto numero di dispositivi impiegati rispetto al mass-trapping tradizionale) risulta ancor più evidente la 

validità del suo impiego. Tuttavia la validità assoluta del metodo va verificata ed ulteriormente sperimentata anche in 

altre realtà olivicole in diverse condizioni ambientali, e, inoltre, va considerata quale utile mezzo di integrazione con 

altri sistemi di lotta “alternativa”, come ad esempio la raccolta effettuata nell’epoca ottimale. 

Circa i principi attivi saggiati (rame, rotenone ed azadiractina), ammessi all’impiego nella coltivazione biologica, essi 

hanno mostrato buona efficacia nel contenimento di B. oleae . Tale efficacia è apparsa evidente contro la popolazione 

adulta, specialmente nel secondo anno di prove con due trattamenti, ma soprattutto contro la popolazione 

91 

% 

80 

60 

40 

20 

0 

3/8 

13/8 

Carolea (2001) 

Trattamento Trattamento 

23/8 

2/9 

12/9 

22/9 

2/10 

Tesi A (Rotenone) 

Tesi B (Azadiractina) 

Tesi C (Controllo) 


12/10 

22/10

preimmaginale. L’infestazione attiva, infatti, nelle tesi trattate e sulle diverse cultivar, si è sempre attestata su valori 

compresi entro il 20%, limite ritenuto valido per l’ottenimento di un prodotto (olio) di qualità. Nel confronto tra 

rotenone e azadiractina, il primo ha mostrato avere una maggiore, sia pur lieve, efficacia rispetto alla azadiractina. 

Quest’ultimo prodotto, indicato come “fagoinibitore” e “regolatore di crescita”, ha in realtà funzionato come 

“insetticida” , visto l’effetto sull’abbattimento della popolazione adulta e la mortalità delle larve riscontrata nell’esame 

dei campioni di olive. Tuttavia nell’ipotesi di un loro più largo impiego in olivicoltura, destano notevole perplessità i 

risultati delle analisi sui residui del prodotto, che per il rotenone figurano in misura eccessiva, tanto nelle olive quanto 

nell’olio. Inoltre, non sono nemmeno noti gli effetti che questi prodotti provocano nell’impatto con l’ecosistema, per i 

quali, vista la loro attività insetticida non selettiva, non è difficile immaginarne una incidenza negativa nei confronti 

dell’artropodofauna utile, soprattutto per l’azadiractina che all’effetto insetticida unisce anche quello di fagoinibitore 

(alterazione delle abitudini alimentari) e di bioregolatore (alterazione ormonale della crescita e della metamorfosi). 

Occorrono ulteriori specifiche prove sperimentali per approfondire le conoscenze al riguardo e, ove si accertassero 

negativi impatti ambientali e si confermassero rischiose presenze di residui nel prodotto, per questi principi attivi si 

imporrebbe una revisione della tabella IIB del REG.CEE 2092/91. 

Bibliografia 

Bagnoli B., Petacchi R., 2001- Problematiche relative all’utilizzo di prodotti di origine vegetale per il controllo della 

mosca delle olive. Pisa, aprile (in press). 

Belcari A., Bobbi E., 1999 – L’impiego del rame nel controllo della mosca delle olive, Bactrocera oleae. Informatore 

fitopatologico, 12: 52-53 

Delrio G., Lentini A., 1993 – Applicazione della tecnica delle catture massali contro il Dacus oleae in due comprensori 

olivicoli dalla Sardegna. Atti del Convegno “Olivicoltura”, Firenze 1991:41-45. 

Haniotakis G., Kozyrakis M., Fitsakis T., Antonidaki A., 1991 – An effective mass trapping method for the control 

of Dacus oleae. J. Econ. Entom. , 84 

Iannotta N., Perri L., Rinaldi R., 1994 - Control of the olive-fly by mass-trapping in Calabria. Acta Horticulturae, n. 

356: 411-413. 

Iannotta N., Monardo D., Perri L., Tocci C., Zaffina F., 2000- Esperienze di lotta alla Bactrocera oleae (Gmel.) con 

sistemi conformi al Reg. CEE 2092/91. Atti Sem. “Metodi e sistemi innovativi dell’olivicoltura biologica e sostenibile” 

, Rende (Cs) :123-126. 

Iannotta N., 2001- La lotta naturale ai parassiti dell’olivo. Olivo e olio n° 5: 16-26. 

Iannotta N., 2001- Esperienze di lotta contro Bactrocera oleae (Gmel) con metodi conformi al Reg. CEE 2092/91. 

Relazione al convegno “L’olivicoltura biologica e la lotta contro la mosca delle olive”. Pisa, aprile (in press). 

Iannotta N., Lombardo N., Maiolo B., Parlati M.V., Scazziota B., 2002 - Controllo di Bactrocera oleae (Gmel.) con 

un prodotto fitosanitario naturale compatibile con la produzione biologica. Atti Conv. “Produzioni alimentari e qualità 

della vita”, 4-8 Sett.2000-Sassari: 333-338. 

Petacchi R., Rizzi I., Guidotti D., Toma M., 2001 – Informatizzazione della raccolta e gestione dei dati nei 

programmi finalizzati al controllo della mosca dell’olivo: l’esperienza della Regione Toscana nella tecnica delle 

“catture massali”, Pisa, aprile (in press). 


92

17 

Il contenimento della ticchiolatura in frutticoltura biologica 

Markus Kelderer 1 , Claudio Casera, Ewald Lardschneider 

Centro Sperimentale Agrario Forestale Laimburg (BZ) 

Spesso i produttori biologici sono obbligati dall’andamento del mercato a scegliere le stesse cultivar coltivate in 

frutticoltura convenzionale ed integrata. La maggior parte di queste sono abbastanza sensibili alla ticchiolatura e 

non è facile arrivare a fine stagione senza gravi danni. La ricerca condotta presso il Centro Sperimentale 

Laimburg dimostra che è possibile controllare la ticchiolatura delle pomacee usando bassi dosaggi di rame, 

polisulfuro di calcio ed argille acide in combinazione con lo zolfo. Il controllo è efficace se questi prodotti 

vengono usati nel giusto modo. 

I punti principali di questo lavoro sperimentale sono: 

a) Confronto e riduzione del dosaggio di diverse sostanze attive alternative al rame 

b) Tempi più appropriati di applicazione delle sostanze attive (preventivo, tempestivo, curativo) 

c) Tecniche di applicazione (atomizzatore, sistemi d’irrigazione per aspersione) 

d) Una particolare attenzione viene posta attualmente agli interventi sanitari per ridurre l’inoculo nel 

frutteto 

a) In cultivar molto sensibili alla ticchiolatura (es. Golden Delicious) l’efficacia delle miscele argille 

acide/zolfo a volte non sono sufficienti, mentre il rame dimostra una eccellente efficacia anche a 

dosaggi molto bassi (150g di rame metallico per trattamento ed ettaro), provocando però su certe varietà 

problemi di rugginosità. Tra le formulazioni a base di zolfo, è da segnalare in particolar modo il 

polisolfuro di calcio. Questo è abbastanza efficace e viene tollerato abbastanza bene anche dalla pianta. 

Da quando questo prodotto è stato introdotto negli allegati 2b del regolamento comunitario 2092/91 la 

maggior parte dei produttori usa questa sostanza attiva in dosaggi che variano da 10 a 30kg per 

trattamento ed ettaro in funzione dell’andamento climatico durante la stagione. 

b) Gli ultimi anni i produttori biologici hanno usato questi fungicidi soprattutto in modo preventivo. Per 

ridurre il numero di trattamenti ed aumentarne l’efficacia, a partire del 1997 al Centro Sperimentale 

Laimburg, sono stati sperimentati sia in laboratorio che in pieno campo, trattamenti tempestivi (scab 

stop) su foglia bagnata e trattamenti curativi. I risultati dei trattamenti tempestivi su foglia bagnata sono 

incoraggianti e i produttori stanno introducendo queste tecniche nei loro frutteti. Il tempo ideale per 

effettuare questi trattamenti è abbastanza breve, perciò è necessario poter disporre di una tecnica di 

applicazione veloce ed efficace. 

c) Una possibile opzione nell’ambito delle tecniche di applicazione e l’uso del sistema di irrigazione per 

aspersione. In diverse prove vennero confrontati metodi di applicazione con atomizzatori e sistemi di 

irrigazione per aspersione. I due metodi di applicazione a parità di dosaggio e principio attivo ad ettaro 

hanno fornito risultati confrontabili. 

d) La quantità di inoculo presente è determinante per il grado di pressione della malattia nel frutteto. 

Ridurre l’inoculo può diventare determinante per riuscire a controllare certe patologie. A partire dal 

2001 sono in atto prove per determinare come possa essere ridotto l’inoculo nei frutteti e qual è l’effetto 

di queste misure preventive sull’incidenza della malattia. 


93

18 

INFLUENCE OF DIFFERENT TREATMENTS ON Cd (II), Cu (II), Pb (II) AND Zn (II) CONTENT IN 

SICILIAN OLIVE OILS 

La Pera Lara, Lo Turco Vincenzo, Lo Curto Simona, Mavrogeni Ekaterini, Dugo Giacomo 

Dipartimento di Chmica Organica e Biologica Università di Messina Salita Sperone, 31, 90166 S. Agata- 

Messina 

Metals can be found in fatty food as edible oils that are often subjected to refining, bleaching, and 

deodorization, during which they inevitably come in contact to metallic surfaces (1). Trace levels of some metal 

ions like Cu (II) could catalyse the oxidation reaction of fatty acid chains, with conseguent a deleterious effect on 

oil flavor and shelf-life of oils (2). 

Metals contamination of olive oil could be due to their presence in the soil, water and, particularly for 

lead, in the air (3). Many studies have been carried out in which the presence of trace metals in olive oils was 

correlated to agronomical techniques, harvesting methods, oil extraction and packaging procedures (4, 5). The 

purpose of this paper was to explore the correlation between the pesticide treatments occurred in olive growing 

and the content of metals found in oils. 

The analytical techniques frequently used for the subsequent determinations are emission and absorption 

spectrophotometric techniques as well as electroanalytical techniques (2,6). 

In this paper derivative potentiometric stripping analysis (dPSA) was used to determine copper (II), lead 

(II), cadmium (II) and zinc (II), in samples of virgin olive oils from Sicily (7-9). 


Samples 

Seven olive oils samples belonging to Nocellara del Belice variety, produced in the crop year 2000 -2001 

from plants grown up on sandy soils in the zone of Valle del Belice (Trapani), were studied. Each of these 

samples was originated from olive grove subjected to different pesticides treatments as dimethoate/fenthion, 

dimethoate, copper oxy-chlorine/dimethoate, copper oxy-chlorine/fenthion; biological treatments as copper oxychlorine 

and biological fight and no treatment. 

Oil samples were stored in dark glassy bottles at 4°C for the time of analysis. 

Chemicals 

Ultra pure hydrochloric acid (34-37%), Hg (II) (1000 µgmL -1 , 1M in hydrochloric acid) and Cd (II), Cu 

(II), Pb (II), Zn (II), Ga (III) and Pd (II) (1000 µg mL -1 , 0.5 N in nitric acid) standard solutions were purchased 

by Panreac Quimica (Barcelona, Spain). The extracts are filtrated on a carbon column Supelclean ENVI-Carb 

SPE (0.5 g, 6 mL), purchased by Supelco (Bellefonte, PA, USA). 

Apparatus 

Determinations were carried out using a PSA ION 3 potentiometric stripping analyzer (Steroglass, S. 

Martino in Campo, Perugia, Italy), connected to an IBM-compatible personal computer. The analyzer operated 

under the control of the NEOTES software package (Steroglass). 

Electrode Preparation (plating) and potentiometric determination 

Working electrode was a glassy carbon electrode coated with a Hg film, the reference one was an 

Ag/AgCl electrode; the counter was a Pt electrode. The plating of the working electrode was effected putting in 

the electrochemical cell a 20 mL volume of a 1000 µgmL -1 Hg standard solution and carrying out the 

electrolysis, at –950 mV against the reference electrode, for 1 min. 

The determination of Cd, Cu and Pb in oily extract was executed at pH 0.5, putting in the electrochemical 

cell a 3 mL volume of elute, a 17 mL volume of ultra pure H2O and a 1 mL volume of Hg (II) standard solution 

as oxidant agent. The determination of Zn was executed at pH 1.8, putting in the electrochemical cell a 1 mL 

volume of elute, 18.5 mL of ultra pure H2O and 0.5 mL of Hg (II) standard solution as oxidant agent. The 

quantitatve analysis was carried out by the multiple standard addition method. Metals determination was 

managed under analytical conditions described in table 1. 

RESULTS AND DISUSSION 

Method 

The repeatability of the method was attested at 86.4 % for cadmium, at 94.9% for copper, at 99.0% for 

lead and at 98.9% for zinc. Recoveries were carried out by spiking a sample of olive oil at different levels. 


94

Obtained recoveries percent were: 84.5±9.9% for Cd, 97.3±2.7% for Cu, 1 00.7±0.7 % for Pb and 83.4±1.7 % for 

Zn. Instrumental detection limits were: 1.2 ngg -1 for Cd, 3.6 ngg -1 for Cu, 5.9 ngg -1 for Pb and 14.3 ngg -1 for Zn. 

Application 

Obtained results are reported on Table 1. Cd (II) was not found in any olive oil sample. The highest 

content of Cu (II) was found in the sample originated from biological strategy (96.1±5.3 ng g -1 ). A lower 

content of the metal was presented by the samples produced from plants treated with organic pesticides 

associated or not to copper oxy-chlorine, such as copper oxy-chlorine/dimethoate (91.4±5.1 ng g -1 ) and copper 

oxy-chlorine/fenthion (60.0±3.1 ng g -1 ). A smaller level of Cu was found in samples originated from 

dimethoate/fenthion (56.8±3.3 ng g -1 ), dimethoate (38.3±0.4 ng g -1 ) and no treated sample (44.8±1.5 ng g -1 ). 

The smallest Cu concentration was found in the oil originated from copper oxy-chlorine treatment (26.0±2.3 ng 

g -1 ). 

Oil samples produced from olive grove treated with organic pesticides - copper oxychlorine/fenthion 

(222.6±2.3 ng g -1 ), dimethoate (179.2 ±1.9 ng g -1 ), dimethoate/fenthion (165.0±1.5 ng g -1 ) - showed a higher 

lead amount respect to samples treated only with copper oxy-chlorine (47.5±0.5 ng g -1 ), no-treated sample 

(34.6±0.4ng g -1 ) and the sample from biological strategy (17.3±0.2 ng g -1 ), which presented the lowest Pb 

content (fig.1). An exception was represented by the sample treated with copper oxy-chlorine/dimethoate 

(32.8±0.3 ng g -1 ). 

Samples produced from olive groves treated with copper oxy-chlorine/fenthion (576.0±6.3 ng g -1 ) 

presented the highest content of Zn, followed by biological strategy (550.0±5.8 ng g -1 ) and copper oxychlorine/dimethoato 

(433.2±4.7 ng g -1 ) samples; the oils treated with dimethoate (176.0 ±1.9 ng g -1 ), copper 

oxy-chlorine (161.5±1.7 ng g -1 ), dimethoate/fenthion (140.8±1.5 ng g -1 ), showed a Zn level remarkably 

lower. The smallest concentration of the metal was found in no-treated sample (59.8±0.7 ng g -1 ). 

CONCLUSION 

Samples produced from no treated olive grove and by plants treated only with copper oxy-chlorine, 

always showed a lower content of Cu (II), Pb (II) and Zn (II) respect to samples originated from olive grove 

treated with the organophosphorated pesticides dimethoate and fenthion. These molecules, which contain S, P 

and O atoms, may influence the mechanism of metals uptake of the plants by complexing the cations (10). 

Samples originated from biological fight always present a high amount of the micronutrient Cu (II) and Zn (II) 

and a very low concentration of Pb (II). 

LITERATURE 

1. Martín-Polvillo M., Albi T. and Guinda A. Determination of trace elements in edible vegetable 

oils by atomic absorption spectrophotometry. J. Am. Oil Chem. Soc. 1994, 71, 347-350. 

2. Calapaj R., Chiricosta S., Saija G. and Bruno E. Method for the determination of heavy metals 

in vegetable oils by graphite furnace atomic absorption. At. Spectrosc. 1988, 9, 107-110. 

3. Paoletti R., Nicosia S., Clementi F., Fumagalli G. 1 st ed. “Tossicologia degli alimenti”, 1999, 

Utet, Torino. 

4. De Leonardis A., Macciola V. and De Felice M. 1997. La determinazione del ferro e del rame 

negli oli vergini d'oliva mediante spettrofotometria ad assorbimento atomico. Riv. Ital. Sostanze Grasse 74: 

149. 

5. Garrido M.D., Frías I., Díaz C. and Hardisson A. Concentrations of metals in vegetable edible 

oils. Food Chem. 1994, 50, 237-240. 

6. Wahdat F., Hinkel S. and Neeb R. Direct inverse voltammetric determination of Pb, Cu and Cd 

in some edible oils after solubilization. Fresenius' Z. Anal. Chem. 1995, 352, 393-396. 

7. La Pera L., Lo Curto S., Dugo G.mo, Lo Coco F. and Liberatori A. Determination of Copper 

(II), lead (II), cadmium (II) and zinc (II) in olive oils from Sicily by derivative potentiometric stripping 

analysis (dPSA). Presented at “European Conference of Advanced Technology for Safe and High Quality of 

Food”, Berlin, 5-7 December, 2001. 

8. La Pera L., Lo Curto S., Visco A., La Torre L. and Dugo G.mo Derivative potentiometric 

stripping analysis (dPSA) used for the determination of cadmium, copper, lead and zinc in Sicilian olive 

oils. J. Agric. Food Chem. 2002, 95, 3090-3093. 

9. La Pera, L.; Lo Coco, F.; Mavrogeni, E; Giuffrida, D.; Dugo, G.mo. Determination of Copper 

(II), Lead (II), Cadmium (II) and Zinc (II) in virgin olive oils produced in Sicily and Apulia by Derivative 

Potentiometric Stripping Analysis. Ital. J. Food Science 2002 in press. 

10. Kaim W.; Schwedersky B. Bioinorganic Chemistry: inorganic elements in the chemistry of 

life. V th ed. Wiley & Sons Ltd, West Sussex, England, 1996. 


95

Table 1. Analytical conditions for determination of Pb, Cu, Cd and Zn. 

Pb Cu Cd Zn 

Integration range mV -580; -380 -380; -140 -760; -590 -1100; -750 

Potential range mV -1200; -100 -1200; -100 -1200; -100 -1200; -100 

Conditioning potential mV 50 x 5 s 50 x 5 s 50 x 5 s 50 x 5 s 

Accumulation potential mV -1200 -1200 -1200 -1200 

Accumulation time min 2 2 2 2 

Stripping time s 10 10 10 10 

Acquisition final potential mV 0 0 0 0 

Sampling time µs 300 300 300 300 

Discharge potential mV -430 -260 -640 -950 

Agitation speed turn/s 2 2 2 2 

Cycles n. 4 4 4 4 

Standard additions n. 2 2 2 2 

Table 2. concentration of Cd, Cu, Pb and Zn in olive oils from different pesticides treatments 

Treatments Cd 

(ng g -1 Cu 

) (ng g -1 Pb 

) (ng g -1 Zn 

) (ng g -1 ) 

Biological fight 

[Pb] (ng/g) 

250,00 

200,00 

150,00 

100,00 

50,00 

0,00 

Figure 1.Pb content found in olive oils from different treatments 

97 

Copper-Fenthion 

Dimethoate-Fenthion 

Dimethoate 

No treatments 

Copper-Dimethoate 

Copper 

Biological fight 


19 

DETERMINATION OF PHENOLIC COMPOUNDS IN EXPERIMENTAL WINES SUBJECTED TO 

DIFFERENT PESTICIDES TREATMENTS 

G. L .La Torre, T. M. Pellicanò, D.Pollicino, M. Alfa, G.mo Dugo 

Università degli Studi di Messina; Dip.to di Chimica Organica e Biolog ica 

INTRODUCTION 

Wine is a complex fluid; it contains water, sugars, acids, alcohols and a wide range of phenolic compounds. The 

phenolics can be derived from grapes and wood, or they can be metabolites from yeasts. Phenolics occur 

naturally in various families of plants, but grapes and related products are considered one of the most important 

dietary sources of these substances (1, 2). Phenolic compounds are an important group of substances that 

contribute to several sensorial characteristics such as colour, flavour, astringency and hardness of wine. 

Furthermore, these compounds are important in food hygiene because of their bactericidal effects and, 

consequently, they constitute an interesting index for evaluating the quality of a wine (3 - 5). Some researchers 

suggest that a regular consumption of a moderate quantity of wine, especially red (6-8), is therapeutic for human 

health. This protective effect has been associated with an increase in the plasma of the level of high-density 

lipoprotein (HDL)-cholesterol and with a decrease of platelet activity, because of the effect of ethanol and 

polyphenolic components (9). Epidemiological studies have shown that a moderate wine consumption helps to 

prevent the development of coronary heart disease (10,11). 

The major molecules responsibles for this physiologic protection are phenolic compounds such as resveratrol 

and flavonoids. Moreover, it has been reported that resveratrol, quercetin and other polyphenolics are cancer 

chemo-preventive agents, since through the inhibition of cyclooxygenase they restrain cellular events associated 

with tumour initiation, promotion and progression, and stimulate quinone reductase – an enzyme that detoxifies 

carcinogens (12,13). In recent years, interest has been focused on resveratrol (3,5,4’-trihydroxystilbene), a 

phytoalexin produced by the plant as a defence response to some exogenous stimuli, such as ultraviolet radiation, 

chemical stressor and, particularly, microbial infections (15). It is well-known that resveratrol is present in the 

chemical form of cis- and trans-resveratrol and of cis- and trans-piceid ( β-glucosides of resveratrol) (16). The 

presence of resveratrol has been detected in numerous types of wines, originating from various countries (USA, 

France, Italy, Spain, Japan), and large variations in its content are reported. They depend on grape cultivar, 

geographic origin, wine type, Botrytis infection and enological practices. Generally, many authors found that 

higher contents of trans-resveratrol (0.2 - 13 mg/L) are usually present in red wines that have had a prolonged 

contact between the must and skins, whereas lower concentrations (0.1 - 0.8 mg/L) are usually present in white 

wine (17, 18). In rosé wines the levels of resveratrol monomers were between white and red wines (18). About 

β-glucosides of resveratrol, no proportionality between the levels of cis- and trans-resveratrol and their 

glucosides were reported (19), whereas piceid concentrations from 0.3 mg/L to 9 mg/L in red wines and from 0.1 

mg/L to 2.2 mg/L in white wines have been detected (14). Studies on resveratrol content in wines are numerous 

(20-24); they are often focussed on the different grape cultivars and associated with the identification of 

polyphenolic compounds, of potential biological activities, such as quercetin, myricetin, etc. The phenolic 

compounds are secondary plant metabolites that are contained within the skin, seed, and flesh of grapes and are 

extracted into wines (especially red) during the process of vinification. The types and concentrations of the 

phenolic compounds in wine may depend on a number of factors: grape variety and ripening stage, soil and 

climatic conditions. Moreover, the processes of viticulture and vinification, which vary between countries, 

regions and wine-makers, determine the content and profile of phenolic compounds in wine (14). The 

concentration of phenolic compounds is marked by temperature, so places with high temperatures and drier 

conditions disfavour the presence of these compounds in wines.(16) 

The purpose of this study was to determine the polyphenolic compounds (resveratrol, piceid, and flavonoids) in 

wines from three different Italian regions, obtained from grapes (Sangiovese, Fiano d’Avellino, Inzolia- 

Caricante (50/50)) treated with different pesticides and to investigate the probable influence of the different 

treatments on the quality of the wines. 


Chemicals and materials 

trans-Resveratrol was purchased from Sigma Chemical Co. Stock solution of cis-resveratrol was produced by UV 

irradiation of trans-resveratrol in methanol for 30 min at 254 nm. Solid-phase extraction (SPE) cartridges were 


98

Supelco Supelclean LC-18 SPE tubes (3ml). The other phenolic compoundswere quercetin, rutin (quercetin-3-Orutinoside), 

kaempferol, kaempferol-3-glucoside, kaempferol-3-rutinoside, rhamnetin, isorhamnetin, and were 

also supplied by Sigma. 

Wine Analysed 

The analyses were carried out on 23 Italian wine samples (red and white), coming from three different Italian 

vineyards: one located in Tuscany, one in Campania and one in Sicily. In particular, wines from Tuscany were 

produced from 30 years old plants (vines were grafted with Sangiovese variety, Morellino clone) and each sample 

was vinified red. Wines from Campania were produced from 20 years old plants (vines were grafted with Fiano 

d’Avellino variety) and each sample was vinified white. Sicilian wines were produced from 15-20 years old 

plants (vines were grafted with Inzolia and Carricante variety in ratio 1:1) and each sample was vinified white. 

The vineyard was subdivided in thesis; each thesis consisted of four rows of vines and the remotest was located 

with the function to prevent drift effect. The fungicides treatments were carried out at the end of blossoming and 

maturation. The scheme of experimental work provided the treatment of each thesis with different antioidics and 

antiperonosporics: sulphur, azoxystrobin, dinocap, penconazolo, fenarimol, quinoxyfen (tab.1). 

Analysis of cis- and trans-resveratrolo 

cis- and trans-resveratrol were extracted from wines using the SPE cartridges prior to the HPLC analysis. For the 

analysis of total wine resveratrol was used an enzymatic hydrolysis method, improved by our working group (25). 

The wine resveratrol O-glycosides (trans- and cis-piceid) were totally hydrolysed in ~ 9 hours after incubation 

with β-glucosidase at 50°C; then the trans- and cis-aglycones were measured by HPLC after solid phase 

extraction (SPE). An ODS Hypersil 5µm (250x4,6 mm i.d.) column was used as the stationary phase and was 

preceded by a precolumn of the same material. The mobile phase consisted of phase (A) water/acetic acid (pH = 

3) and phase (B) acetonitrile/acetic acid (pH = 3). cis- and trans-resveratrol were eluted with a gradient time 

program and the eluted was monitored at 285 and 307 nm, where cis- and trans- isomers have absorbance 

maxima, respectively. All chromatographic experiments were performed at room temperature with a flow rate 

1ml/min. The chromatograms were recorded according to the retention time. Before the HPLC analysis the 

extract were filtered through a 0,45µm glass-microfiber GMF Whatman chromatographic filter. 

HPLC analysis methodology for flavonoids 

The analysis were carried out on aliquot of wine filtered through a 0,45 µm nylon Supelco chromatographic filter, 

using an HPLC system operating at room temperature with a flow–rate of 0,8 ml/min.. The spectrophotometric 

detector was a photodiode array operating at wavelengths of 265 nm for aglycon-flavonoids and 254 for 

flavonoid-glucoside. The analytical column was a Alltech Alltime C-18, 5 µm ( 250 x 4,6 mm i.d.). A SUPELCO 

guard column packed with the same stationary phase was also used. The mobile phase consisted of 

H2O/CH3COOH at pH= 3 (phase A) and CH3CN/CH3COOH at pH = 3 (phase B). Elution was carried out in 

gradient. 


Analytical data related to the free isomeric forms of resveratrol (cis- and trans-) and to the total resveratrol (free 

and glucosides isomers) are reported in fig. 1,2 and 3. This investigation, once again, clearly shows that red wines 

have a greater resveratrol content than white wines, regardless of the pesticide treatments of the vines. Analysing 

the data related to wines from Campania, it can be noticed that in the samples Thesis 3, 4, 5 and 6 the amount of 

cis-resveratrol is between 0.04 and 0.05 mg/L, while the samples Thesis 1, Thesis 2 and Thesis 7 have an amount 

of cis-resveratrol impossible to verify with the adopted analytic method. The concentration of trans-resveratrol 

results highest in the sample Thesis 5 (0.13 mg/L), and a lowest in the sample Thesis 3 (0.07 mg/L). The total 

concentration of cis- and trans-resveratrol deserve a particular attention because, generally, it is similar in every 

sample while, this balance does not occur in free form of cis- and trans-resveratrol. In fact, the concentration of 

free trans-resveratrol is a little lower than that of total trans-resveratrol, but in sample Thesis 2 it is even almost 

identical; the same situation does not occur in cis-resveratrol. Examining the concentration of free cis-resveratrol 

to that of total, it results that the amount of cis-piceid is much higher than that of trans-piceid, regardless of the 

pesticides treatment. A completely different situation is shown for wines produced and vinified in Sicily. In this 

case, the concentration of total trans-resveratrol (between 0.17 and 0.34 mg/L) is always higher than that of total 

cis-resveratrol (between 0.09 and 0.20 mg/L). Moreover, differently from what was noticed in wines produced 

and vinified in Campania, the cis- and trans-piceid concentration in each sample is comparable, and is between a 

minimum value of 0.03 mg/L and a maximum of 0.18 mg/L. The samples Thesis 4 and Thesis 5 differ from this 

general trend, as they are characterized by an amount of cis-piceid clearly higher than that of trans-isomer. As 

shown in fig.3, the wines obtained from cv Sangiovese have very different quantities of free trans-resveratrol and 

they range from 0.22 to 1.76 mg/L. The same trend can be noticed with free cis-resveratrol, but in this case the 

concentrations are more homogeneous. Comparing total cis- and trans-resveratrol concentrations to those of the 

equivalent free isomers, it emerges that the levels of trans-piceid are approximately twice as high as those of the 

corresponding cis-piceid, except for samples Thesis 4 and Thesis 5 that present comparable levels. The 


99

investigation of the flavonoids regarded the identification and quantification of seven components (quercetin, 

rutin, kaempferol, kaempferol-3-glucoside, kaempferol-3-rutinoside, rhamnetin, isorhamnetin) through a fast 

method that involves the use of wine as it is, as suggested by Viñas et al. (14). 

The wine samples from Campania are characterized by a higher content of flavonoids and flavonoid-glucosides 

and show a composition, both qualitatively and quantitatively, similar. Of the seven compounds analysed in the 

flavonoids group, quercetin (

12. Agullo, G.; Gamet, L.; Besson, C.; Domigne, C.; Remesy, C. Quercetin exerts a preferential cytotoxic 

effect on active dividing colon carcinoma HT29 and Caco-2-cells. Cancer Letter 1994, 87, 55-63. 

13. Jang, M.; Pezzuto, J. M. Cancer chemopreventive activity of resveratrol. Drugs Experiments and Clinical 

Research 1999, 213, 65-77. 

14. Viñas, P.; Lòpez-erroz, C.; Marin-Hernàndez, J. J.; Hernàndez-Cordoba, M. Determination of phenols in 

wines by liquid chromatography with photodiode array and fluorescence detections. J. Chrom. A 2000, 

871, 85-93. 

15. Langcake, P.; Pryce, R. J. The production of resveratrol by “Vitis Vinifera” and other members of the 

“Vitacee” as a response to infection or injury. Physiology and Plant Pathology 1976, 9, 77-86. 

16. Goldberg, D. M.; Yan, J.; Ng, E.; Diamandis, E. P.; Karumanchiri, A.; Soleas, G.; Waterhouse, A. L. A 

global survey of trans-resveratrol concentrations in commercial wines. Am J. Enol. Vitic. 1995, 46 (2), 

159-165. 

17. Dugo, G.mo; Bambara, G.; Salvo, F.; Saitta, M.; Lo Curto, S. Contenuto di resveratrolo in vini rossi da 

uve alloctone e autoctone prodotte in Sicilia. L’enologo 2000, 12, 79-84. 

18. Romero-Pérez, A. I.; Lamuela-Raventòs, R.; Waterhouse, A. L. ; De La Torre-Boronat, M. C. Level of cis- 

and trans-Resveratrol and their glucosides in white and rosè Vitis Vinifera Wines from Spain. J. Agric. 

Food Chem. 1996, 44, 2124-2128. 

19. Waterhouse, A. L.; Lamuela-Raventòs, R. The occurrence of piceid, a stilbene glucoside in grape berries. 

Phytochemistry. 1994, 12, 571-573. 

20. Vrhovsek, U.; Wendelin, S.; Eder, R. Effectes of various vinification techniques on the concentration of 

cis- and trans-resveratrol and resveratrol glucoside isomers in wine. Am.J.Enol.Vitic. 1997, 48 (2), 214- 

219. 

21. Nevado, J. J. B.; Salcedo, A. M. C.; Penavo, G. C. Simultaneous determination of cis-and trans-resveratrol 

in wines by capillary zone electrophoresis. Analyst 1999, 124, 61-66. 

22. Sato, M.; Suzuki, Y.; Okutsuka, K. Contents of resveratrol, piceid, and their isomers in commercially 

available wines made from grapes cultivated in Japan. Biosci., Biotecnol., Biochem. 1997, 61 (11), 1800- 

1805. 

23. Chu, Q.; O’Dwyer, M.; Zeele M. G. Directed analysis of resveratrol in wine by micellar electrokinetic 

capillary electrophoresis. J. Agric. Food Chem. 1998, 46, 509-513. 

24. Ribeiro de Lima, M.T.; Waffo-Te’guo, P.; Teissendre, P.L.; Pujolas, A.; Vercauteren,J.; Carbanis, J. C.; 

Merillon, J. M. Determination of stilbenes (trans-Astringin, cis- and trans-Piceid, and cis- and trans- 

Resveratrol) in Portuguese Wines. J. Agric. Food Chem. 1999, 47, 2666-2670. 

25. La Torre, G. L.; Laganà, G.; Bellocco, E.; Vilasi, F.; Salvo, F.; Dugo, G.mo Enzymatic method for 

analysis of resveratrol glucosides in wine. Anal.Chim.Acta (Submitted). 


101

Table 1 Scheme of experimental treatment of each wine sample with antioidium and antiperonospora 

Wine Sample Pesticides n° treatments Pesticides n° treatments 

Campania 

Thesis 1 Powdered sulphur 2 Quinoxyfen 6 

Thesis 2 Powdered sulphur 2 Fenarimol 6 

Thesis 3 Powdered sulphur 2 Azoxystrobin 6 

Thesis 4 Dinocap 2 Penconazole 6 

Thesis 5 Wettable sulphur 2 Wettable sulphur 6 

Thesis 6 Wettable sulphur 2 Dinocap 6 

Thesis 7 Powdered sulphur 2 Powdered sulphur 6 

Sicily 






Thesis 6 Wettable sulphur 2 Dinocap 6 


Tuscany 






Thesis 6 Dinocap 2 Sulphur + Quinoxyfen 6 + 3 


Table 2 - Detection limits for the analyses of flavonoids 

Wine 

Sample 

Rutin Kaempferol- 

3-Rutinoside 

Kaempferol- 

3-Glucoside 

Kaempferol Quercetin Isorhamnetin Rhamnetin 

Thesis 1 2.72 < 0.64 2.54 < 0.25 7.52 4.16 10.02 

Thesis 2 2.79 < 0.64 2.58 < 0.25 7.52 4.16 10.02 

Thesis 3 2.72 < 0.64 2.57 < 0.25 7.58 4.17 10.03 

Thesis 4 2.72 < 0.64 2.56 < 0.25 7.59 4.17 10.02 

Thesis 5 2.72 < 0.64 2.57 < 0.25 7.53 4.17 10.02 

Thesis 6 2.72 < 0.64 2.56 < 0.25 7.52 4.16 10.02 

Thesis 7 2.72 < 0.64 2.46 < 0.25 7.51 4.16 10.03 


102

Tab. 3 Flavonol content (µg/ml) in white wines from Campania 

Flavonoid µg/ml 

Rutin 0.03 

Kaempferol 0.25 

Kaempferol-3-glucoside 0.51 

Kaempferol-3-rutinoside 0.64 

Quercetin 0.52 

Rhamnetin 0.55 

Isorhamnetin 0.24 

Table 4 Flavonol content (µg/ml) in white wines from Sicily 

Wine Sample Rutin 

Kaempferol- 

3-Rutinoside 

Kaempferol- 

3-Glucoside 

Table 5 Flavonol content (µg/ml) in red wines from Tuscany 

103 


Thesis 1 2.72 < 0.64 2.41 < 0.25 < 0.52 < 0.24 10.06 

Thesis 2 2.90 < 0.64 2.39 < 0.25 < 0.52 4.20 10.02 

Thesis 3 2.85 < 0.64 2.39 4.29 < 0.52 4.17 10.03 

Thesis 4 2.84 < 0.64 2.56 4.29 7.51 4.17 10.02 

Thesis 5 2.88 < 0.64 2.39 4.29 < 0.52 4.17 9.98 

Thesis 6 2.74 < 0.64 2.38 4.29 < 0.52 4.16 10.02 

Thesis 7 2.85 < 0.64 2.38 4.29 < 0.52 4.17 10.02 

Wine 

Sample 

Rutin 

Kaempferol- 

3-Rutinoside 

Kaempferol- 

3-Glucoside 


Thesis 1 13.04 48.95 < 0.51 < 0.25 24.67 < 0.24 < 0.55 

Thesis 2 13.67 3.58 5.68 < 0.25 60.15 5.93 < 0.55 

Thesis 3 19.45 26.48 6.37 < 0.25 21.36 < 0.24 < 0.55 

Thesis 4 22.08 29.55 < 0.51 < 0.25 27.47 < 0.24 < 0.55 

Thesis 5 13.91 38.55 4.35 < 0.25 29.61 < 0.24 < 0.55 

Thesis 6 14.83 44.32 < 0.51 < 0.25 40.15 < 0.24 < 0.55 

Thesis 7 12.87 39.02 < 0.51 < 0.25 20.11 < 0.24 < 0.55 


mg/L 

mg/L 

Fig.1 

0.36 

0.28 

0.21 

0.14 

0.07 

0.00 

0.17 

0.14 

0.11 

0.08 

0.05 

0.02 

0.00 

Fig. 2 

CONCENTRATION OF RESVERATROL ISOMERS 

IN WHITE WINES FROM SICILY 

Thesis 1 Thesis 2 Thesis 3 Thesis 4 Thesis 5 Thesis 6 Thesis 7 


IN WHITE WINES FROM CAMPANIA 


Samples 

Samples 


104 

trans-resveratrol 

trans-piceid 

total transresveratrol 

cis-resveratrol 

cis-piceid 

total cisresveratrol 

trans- resveratrol 

trans-piceid 

total trans- resveratrol 


cis-piceid 

total cis-resveratrol

mg/L 

Fig.3 

3,50 

3,00 

2,50 

2,00 

1,50 

1,00 

0,50 

0,00 


IN RED WINES FROM TUSCANY 


samples 


105 

trans-resveratrol 

trans-piceid 

total trans-resveratrol 


cis-piceid 

total cis-resveratrol

20 

CONTROL OF APPLE SCAB IN ORGANIC FARMING 

Anna La Torre, Lucia Donnarumma, Daniele Lolletti, Giancarlo Imbrogli ni 

Istituto Sperimentale per la Patologia Vegetale – Via C. G. Bartero, 22 - Roma 

Introduction 

Apple scab, which is caused by Venturia inequalis (Cke.) Wint., is considered the mayor disease of apple. In 

organic agriculture, formulations of synthetic origin for defence are not admitted and plant protection is 

essentially based on the use of mineral products as copper and sulphur. Copper is more effective than sulphur 

(sulphur is ineffective at below 10°C, phytotoxic over 28°C and it has greater persistence). Use of copper 

involves several problems: it accumulates in soil, causes phytotoxicity phenomena, when employed on full 

vegetation, it gives rust phenomena on some varieties of apples. It is necessary to find protection alternatives 

strategies, limiting the negative effects of use formulations of this element and assuring, at the same time, 

sufficient protection against the diseases. 

Fig. 1: - Spilocea pomi conidia Fig. 2: - Experimental field 


The trial has been done in a farm near Latina (Latium, central Italy), which as been converted, in 1993, to the 

method of organic farming (cv Royal Gala grafted on M9). Treatments and doses are indicated in table 1. Some 

compounds, for their formulation, release low amounts of copper. They are: Cuivrol, commercialized like 

fertiliser (18% of copper metal); Cuprobenton, composed by copper oxychloride and copper sulfate hydrate 

(15% of copper metal) coformulated with benthic clay (70%); Blue Copper Formula 2, made up with copper 

hydroxide (24% in shape of crystals of the dimension of 0,35 mµ). As alternative products have been tested 

Myco-sin (sulphureous clay, yeasts, equisetum extracts) and calcium polysulphide. The trials have been 

conducted as recommended in EPPO/OEPP PP 1/5(3). The esteem of the infectious risk has been assessed on 

the Mills criteria, monitoring instrumentally the climatic parameters by a thermoigrograph and a pluviometer. 

The assessments to estimate therapeutic efficacy have been carried out on 4 central plants of every parcel 

estimating the number of leaves for every treatment and percentage of leaves surface interested by apple scab, 

using a scale with 5 classes of attack. Results obtained, after arc sin trasformation, have been evaluated by 

Duncan test. 


106

Table 1 – Fungicides used during the trial 

Active ingredient Commercial 

Formula a.s. % o Dose f. c. Dose a.s. Dose Cu 

formula 

g/l ml - g/hl g/hl 

++ 

g/hl 

Copper oxycloride Copper Pro 50 Water-dispersible 50 700; 600; 400; 350; 300; 350; 300; 

WDG 

granules 

340; 288; 200; 200; 170; 200; 170; 

100 

144; 100; 144; 100, 

(WDG) 

50 50 

Copper oxycloride, 

Cuprobenton DC Wettable powder 25 1100; 500 275; 125 165;75 

Copper sulfate hydrate 

5 

55; 25 

Copper hydroxide Rame Azzurro Suspension 350 220; 160 77, 56 52,8; 38,4 

Formula 2 concentrate 

Copper metal with B, Mo, Zn Cuivrol Wettable powder 18 700; 300; 270 126; 54; 126;54; 

48,6 48,6 

Calcium polysulphide Polisenio Liquid 80 18000; 1800;1500 14400; 

1440; 

1200 

Sulphureous clay, leavening Myco-sin* Wettable powder 1500; 1000; 

117; 60,8 

substances,equisetum extracts 

700; 500 

*The treatments have been done in accordance with Biagra company: 

at phenologycal phase “leaf fall” one treatment has been done with Bordeaux mixture (9kg/ha); at “beginning of bud swelling”, “bud 

burst” and “flowering” treatment with Myco-sin and wettable sulphur; at “bud swelling” “pre-flowering” and “fruit swelling” 

treatment with Myco-sin and sea-weed; at “fruit setting” one treatment has been done with copper sulfate (4 kg/ha). 

Results 

Table 2 shows the development of the infections during the trial and the activity of the various formulations over time. 

Table 2 – Percentage of infected leaves 

Treatments 

Assessment 

12/04/00 

Assessment 

18/05/00 

% infect leaves 

Assessment 

20/06/00 

Assessment 

20/07/00 

Assessment 

16/08/00 

Copper oxycloride 3.5 aA 15.3 aA 25.8 aAB 30.4 aA 33.9 aA 


Copper sulfate 

hydrate 

4.3 aA 26.7 abAB 33.1 abABC 52.2 bB 53.3 bcBC 

Copper hydroxide 4.5 aA 17.1 aA 22.9 aA 38.3 aA 43.1 abAB 

Copper metal with B, 

Mo, Zn 

10.2 aAB 41.7 cBC 46.9 bcABC 61.9 cB 72.5 dD 

Calcium 

polysulphide 

1.0 aA 34.7 bcABC 45.1 bcABC 56.3 bcB 63.8 cdCD 

Sulphureous 

clay, leavening 

substances,equisetum 

extracts 

7.5 aA 46.9 cCD 48.6 bcBC 61.3 bcB 65.6 cdCD 

Control 23.1 bB 61.0 dD 51.9 cC 63.4 cB 68.5 dCD 

Different letters indicate significant different values by Duncan test (for P=0,05, lower-case letters, and for 

P=0,01 capital letters) 


107

Temperature (°C) Relative Umidity (%) 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

Fig. 3 reports the assessment related to the degree of infection found on the leaves. On the fruits the first 

symptoms appeared on 11 May; results on the degree of protection offered by different products are reported in 

table 3. During the trial, the climatic conditions have turned out favourable for the development of pathogen 

(Fig. 4). Elevate infection degrees, found on the leaves and the fruits, caused low yields. 

INFECT LEAF INDEX (%) 

50 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

Fig. 3 - Percentage of leaf damage at different date for various products 

CC 

a 

a 

a 

CU 

a 

LEGENDA: = infections of apple scab 

RG= Bud burst OT= Mouse-ear stage MA= Appearance of flower buds BR= Pink bud PF= Full flowering 

CP= Petal fall AL=Fruit setting FN= Walnut fruit IF= Fruit swelling 

108 

a 

a 

b 

a 

a 

b 

IR 

a 

b 

a 

b 

a 

a 

CV 

THESES 

Different letters indicate significant different value by Duncan test for P=0,05 

a 

a 

b 

c 

d 

c 

a 

PS 

c 

d 

b 

c 

b 

c 

a 

M+S 

b 

c 

d 

c 

c 

d 

b 

TEST 

d d d 

e 

c 

18/05/00 

12/04/00 

16/08/00 

20/07/00 

20/06/00 

DATE 

OF ASSESSMENT 

Fig. 4 -Climate during the trial: directions about phenologycal phases and about infections of apple scab 

Infections: 

March April May June 

JuLy Phenologic 

phases: RG OT MA BR PF CP AL FN IF 


LEGEND 

CC = Copper 

oxicloride 

CU = Copper 

oxicloride, Copper 

sulphate hydrate 

IR = Copper hydroxide 

CV = Copper metal 

with B, Mo, Zn 

PS = Calcium 

polysulphide 

M+S = Sulphureous 


substances, 

equisetum extracts 

TEST = Control 

Rain 

30 

25 

20 

15 

10 

5 

0 

Rain (mm) 

Mean temperature 

Mean relative humidity

Table 3 – Efficacy of different treatment against apple scab on fruits (Scale OEPP/EPPO) 

Treatments 

% 

no attack 

Assessment 

20/07/00 

% Fruits with 

1-3 spots 

109 

% Fruits with 

more than 3 

spots 

% 

no attack 

Assessment 

18/08/00 

% Fruits with 

1-3 spots 

% Fruits with 

more than 3 spots 

Copper oxycloride 61.7 eD 26.2 aAB 9.6 aA 70.1 cD 18.9 aA 4.9 aA 


Copper sulfate 

hydrate 

38.0 dC 39.0 cB 27.8 bB 55.3 bC 28.6 bAB 17.6 abABC 

Copper hydroxide 58.5 eD 28.7 abAB 11.7 aA 69.0 cD 19.6 aA 6.9 aAB 

Copper metal with B, 

Mo, Zn 

27.6 cBC 38.2 bcB 38.8 cB 48.8 bBC 33.0 bB 21.0 bcABC 

Calcium polysulphide 24.7 bcB 37.8 bcB 41.7 cBC 46.5 bBC 32.5 bB 24.3 bcBC 

Sulphureous 


substances,equisetum 

extracts 

18.1 bB 27.1 aAB 56.3 dCD 36.2 aAB 35.6 bB 32.6 cCD 

Control 6.1 aA 20.2 aA 68.2 eE 29.2 aA 28.3 bAB 46.1 dD 

Different letters indicate significant different values by Duncan test (for P=0,05, lower-case letters, and for 

P=0,01 capital letters) 

Conclusions 

By the results obtained in the operating conditions of the trial, we can deduce that the various formulates have 

against apple scab an acceptable activity related to the degree of attack found in the control parcels. Best results 

have been obtained with copper oxicloride, reference product in our trials, and also copper hydroxicloride; 

second best results were obtained with the use of Cuprobenton (copper compounds and active bentonites). 

Calcium polysulphide has given partial protection, however, it is a positive result because its employment, also 

after grown resumption, is permitted by Reg. CE n. 1073/2000. Cuivrol, constituted from oligoelements B, Cu, 

Mo, Zn and Myco-sin, made up of sulphureous clay were less effective. Activity against apple scab, not always 

satisfactory, manifested by several products, could in part be related to the strong pressure of the pathogen that 

has found favourable climatic conditions for the insurgence and spread of the disease. 

References 

− Boschieri S., Mantinger H.,1991. Produzione biologico-organica del melo presso il centro per la 

sperimentazione di Laimburg. Otto anni di esperienze. L’informatore Agrario, 44: 97-101. 

− C.M.I. Descriptions of Pathogenic Fungi and Bacteria No.401,1974, Kew. 


− Direttiva del Consiglio 91/414/CEE relativa all’immissione in commercio dei prodotti fitosanitari (G.U. 

19.8.1991, n. L 230). 

− Donnarumma L., La Torre A., 2000. Sali di rame in agricoltura biologica e possibili alternative. 


− Gentili G., Ravagli S., 1994. Applicazione di tecniche di lotta guidata alle malattie crittogamiche dei 

fruttiferi e della vite in Emilia- Romagna: risultati delle sperimentazioni effettuate nel periodo 1986- 

1993. Atti Giornate Fitopatologiche,3: 79-86. 

− Govi G., 1955. Risultati di prove di lotta contro la ticchiolatura del melo. Frutticoltura VI: 517-531. 

− McKinney H.H.,1923. Influence of soil temperature and moisture on infection of wheat seedlings by 

Helminthosporium sativum. Journal of Agricultural Research, 5: 195-217. 

− Mills W. D., 1944. Efficient use of sulfur dust and sprays during rain to control apple scab. Ext. Bull. 

Cornell Agric. Exp. Sta., 630. 

− OEPP/EPPO, 1997. Directives pour l’evaluation biologique des produits phytosanitaires, vol. 2 

Fongicides & Bactericides, PP 1/5(3): 15-18. 


110

21 

CONTAMINAZIONE DA MICOTOSSINE IN ALIMENTI DI PRODUZIONE BIOLOGICA 

A. Maietti 1 , L. Mazzotta 1 , C. Saletti 2 , G. Mirolo 3 , M. Berveglieri 2 , 

P. Tedeschi 1 , V. Brandolini 1 

1Dipartimento di Scienze Farmaceutiche – Università di Ferrara; 2 Dipartimento di Sanità Pubblica – Azienda 

USL di Ferrara; 3 A.R.P.A. – Ferrara 

INTRODUZIONE 

Le micotossine sono metaboliti secondari prodotti da muffe, tossici per gli animali superiori. La formazione di 

queste sostanze è strettamente connessa alla crescita fungina anche se la presenza di funghi tossigeni in un 

prodotto non indica automaticamente la presenza di micotossine. Le tossine tuttavia possono persistere per lungo 

tempo dopo la crescita vegetativa e la morte del fungo. L’analisi micologica basata sulla numerazione delle unità 

vitali e l'identificazione delle specie fungine non permette di quantificare il rischio tossico proprio di un prodotto 

alimentare; questo rischio non può che essere determinato con un'analisi chimico-fisica. 

Le aflatossine sono un gruppo di micotossine prodotte da ceppi di Aspergillus flavus, A. parasiticus e A. nomius, 

con simile struttura chimica. Si ritiene che l'A. flavus produca le aflatossine B1 e B2, mentre l'A. parasiticus 

produca le aflatossine B1, B2, G1, G2. Le contaminazioni degli alimenti con aflatossine si verificano 

principalmente nelle zone a climi caldi e umidi come le zone tropicali e subtropicali, ma poiché i Paesi delle 

zone climatiche più fredde importano prodotti da queste aree, le aflatossine sono un problema mondialmente 

riconosciuto. L'aflatossina B1 è il tipo più frequentemente riscontrato, mentre la presenza di aflatossine B2, G1, 

G2 è generalmente associata alla presenza dell'aflatossina B1 e di solito sono presenti in quantità minore. 

Le muffe capaci di produrre micotossine sono contaminanti assai diffusi negli alimenti e nei prodotti agricoli; 

crescita e produzione di tossine possono avvenire sia in campo che in magazzino. 

Le derrate alimentari possono subire una contaminazione diretta che avviene normalmente su matrici vegetali 

quali cereali, frutta secca, semi oleaginosi, spezie; oppure indiretta attraverso l’alimentazione degli animali da 

allevamento con foraggi e mangimi contaminati. 

La qualità delle materie prime, il controllo dell'ambiente di conservazione, i trattamenti fisici e chimici, la pulizia 

dei sili e dei trasporti, sono la chiave del controllo dello sviluppo fungino. La prevenzione della contaminazione 

fungina potrebbe essere pertanto più difficoltosa nei prodotti biologici che per definizione possono essere 

sottoposti a trattamenti solo con prodotti naturali consentiti. Le micotossine possono causare vari effetti tossici di 

tipo acuto, subacuto, teratogeno, mutageno, cancerogeno; esse evidenziano diversi tipi di tossicità in dipendenza 

della dose, dell'organo interessato, del sesso, dell'età e della specie. 

Per l'Italia, le micotossine rappresentano un problema connesso con l'importazione di derrate da Paesi a clima 

caldo e umido, mentre la contaminazione dei prodotti locali è poco frequente ed è a livelli piuttosto contenuti sia 

per motivi climatici che per le migliori tecniche agronomiche, di raccolta e di conservazione dei prodotti stessi. 

La Comunità Economica Europea con il Regolamento CEE n.1525/98 ha fissato dei limiti massimi accettabili 

sia per il contenuto globale delle aflatossine (4.0 µg/Kg) che per quello della sola aflatossina B1 (2.0 µg/Kg) e 

del suo metabolita aflatossina M1 nel latte (0.05 µg/Kg). 

Il consumo di prodotti da agricoltura biologica è in espansione ed è pertanto maggiore la quota di popolazione a 

rischio con particolare esposizione verso il settore ortofrutticolo (40%) seguito da pasta e riso (8%). Per tale 

motivo si è ritenuto interessante svolgere un'indagine mirata alla valutazione della eventuale presenza di 

micotossine (Aflatossina B1 e totali), nei prodotti "biologici" commercializzati nella provincia di Ferrara al fine 

di incrementare le necessarie informazioni sotto il profilo della sicurezza alimentare. 

Le principali tipologie produttive dell'agricoltura biologica sulle quali si è incentrata la ricerca di micotossine 

sono state le seguenti: cereali e paste alimentari, Muesli biologico, pane integrale biologico; riso integrale 

biologico e riso soffiato biologico, farine biologiche, fette biscottate, crackers, gallette ed affini, e biscotti, 

composta di frutta biologica. 

MATERIALI E METODI 

I campionamenti sono stati effettuati presso la grande distribuzione nel rispetto della normativa vigente ed i 

campioni raccolti sono stati analizzati per verificare la presenza di Aflatossine B1 e totali. 

Procedimento di estrazione: 25 g di campione macinato ed omogeneizzato sono mescolati a 10 g di celite e 

dispersi in una miscela acetonitrile/acido acetico. Dopo 30 minuti di agitazione si filtra. Il filtrato viene trattato 

inizialmente con acqua, piombo acetato neutro e ammonio solfato, e successivamente estratto con toluene. La 


111

fase organica è portata a secco ed il residuo ripreso con esano e acido trifluoroacetico, quindi riportato 

nuovamente a secco e ripreso con acetonitrile e acqua. 

La determinazione analitica è effettuata con un HPLC Perkin Elmer dotato di detector fluorimetrico SFM 25 

Kontron, iniettore Rehodyne con loop da 20 µl e colonna C18 (5 µm x 25 cm). La fase eluente è una soluzione 

costituita da acqua 79 %, acido acetico 7 %, alcol isopropilico 7 % e acetonitrile 7 %. 


I prodotti esaminati in questa ricerca sono tra quelli maggiormente ricercati dal consumatore nell’ambito dei 

prodotti derivati da agricoltura biologica. In questo lavoro sono presentati i dati riguardanti frutta secca, cereali e 

legumi, semi oleaginosi, spezie ed erbe infusionali realtivi a campionamenti effettuati negli anni 1999 e 2000. 

Nell’anno 1999 sono stati esaminati 82 campioni di frutta secca, cereali e legumi; 413 campioni di semi 

oleaginosi; 65 campioni di spezie ed erbe infusionali. Nell’anno 2000 sono stati esaminati 137 campioni di frutta 

secca, cereali e legumi; 430 campioni di semi oleaginosi; 29 campioni di spezie ed erbe infusionali. 

Ricerca: 

Aflatossine 

B1 

B1+B2+G1+G2 

Tot. 

camp 

LR* = limite di rilevabilità 

Ricerca: 

Aflatossine 

B1 

Campioni 

non 

Conformi 

3,7 % 

B1+B2+G1+G2 

FRUTTA SECCA, CEREALI E LEGUMI 

Anno 1999 

Camp 

pos. 

Numero di campioni 

< LR* 0-2 2-4 4-10 10-50 > 50 

112 

Standard di 

riferimento 

(Circolare n.10 

del 9/6/99) 

82 3 75 4 1 1 1 2 µg/kg 

82 3 74 4 1 1 1 1 4 µg/kg 

Tot. 

camp 

B1 


Camp 

pos. 

Campioni 

Conformi 

96,3 % 

137 1 135 1 

Campioni non conformi 

Anno 2000 

Campioni 

non 

Conformi 

3,7 % 

(B1 + B2 + G1 + G2) 

Campioni 

Conformi 

96,3 % 



riferimento 

< LR* 0-2 2-4 4-10 10-50 > 50 (Circolare n.10 

del 9/6/99) 

1 2 µg/kg 

121 1 119 1 4 µg/kg 


Ricerca: 

Aflatossine 

B1 

B1+B2+G1+G2 

B1 

Tot 

camp 


Ricerca: 

Aflatossine 

B1 

Campioni 

non 

Conformi 

0,7 % 

Campioni 

non 

Conformi 

5,6 % 

B1+B2+G1+G2 

Camp 

pos 


SEMI OLEAGINOSI 

Anno 1999 

113 


(B1 + B2 + G1 + G2) 

< LR* 0-2 2-4 4-10 10-50 > 50 


riferimento 


del 9/6/99) 

413 23 387 3 5 7 6 5 2 µg/kg 

413 20 383 6 4 7 7 6 4 µg/kg 

Tot 

camp 

B1 


Camp 

pos 

Campioni 

Conformi 

99,3 % 


Campioni 

Conformi 

94,4 % 

Anno 2000 

Campioni 

non 

Conformi 

0,8% 

Campioni 

non 

Conformi 

4,8 % 


(B1 + B2 + G1 + G2) 

< LR* 0-2 2-4 4-10 10-50 > 50 


riferimento 


del 9/6/99) 

430 18 408 4 5 2 5 6 2 µg/kg 

430 15 408 4 3 4 5 6 4 µg/kg 

Campioni 

Conformi 

99,2 % 

Campioni 

Conformi 

95,2 % 


Ricerca: 

Aflatossine 

B1 

B1+B2+G1+G2 

B1 

Tot 

camp 


Ricerca: 

Aflatossine 

B1 

Campioni 

non 

Conformi 

4,2 % 

Campioni 

non 

Conformi 

3,1 % 

Camp 

pos 


SPEZIE ED ERBE INFUSIONALI 

Anno 1999 

Numero totale di campioni 

< LR* 0-10 10-20 20-50 > 50 

114 

(B1 + B2 + G1 + G2) 


riferimento 


del 9/6/99) 

65 2 53 10 2 10 µg/kg 

65 2 53 10 1 1 20 µg/kg 

B1 

Tot 

camp 

Camp 

pos 


Anno 2000 


(B1 + B2 + G1 + G2) 

< LR* 0-4 4-8 8-10 10- 0 > 50 


riferimento 


del 9/6/99) 

29 1 26 2 1 10 µg/kg 

B1+B2+G1+G2 29 1 26 2 1 20 µg/kg 


Campioni 

Conformi 

95,8 % 

Campioni 

Conformi 

96,9 % 

Campioni 

non 

Conformi 

3,5 % 

Campioni 

non 

Conformi 

3,1 % 

Campioni 

Conformi 

96.5 % 

Campioni 

Conformi 

96,9 % 


Campioni 

non 

Conformi 

3,4 % 

B1 


Campioni 

Conformi 

96,6 % 

115 

Campioni 

non 

Conformi 

3,4 % 

(B1 + B2 + G1 + G2) 

Campioni 

Conformi 

96,6 % 

Relativamente all’anno 1999 a seconda della matrice considerata il grado di contaminazione da aflatossina B1 e 

aflatossine totali è risultato variare dal 3 % nelle erbe infusionali e spezie, al 3,7 % nella frutta secca, al 6 % nei 

semi oleaginosi, mentre nei cereali e legumi analizzati non sono state reperite aflatossine. 

Il trend si è mantenuto nell’anno 2000 per quanto riguarda spezie ed erbe infusionali; per i semi oleaginosi è 

stata riscontrata una maggior percentuale di campioni conformi ai limiti indicati dalla circolare n. 10 del 9/6/99. 

Per la categoria frutta secca cereale e legumi i campioni contaminati da aflatossine sono risultati al di sotto del 1 

%. Tale risultato è ancor più significativo se si considera che per questa categoria di prodotti il numero di 

campioni analizzati è stato circa il doppio. 

All'interno delle diverse classi alimentari sono state individuate alcune matrici più a rischio: tra i semi oleaginosi 

i pistacchi e le arachidi; tra le spezie i peperoncini e le polveri derivate ed infine tra la frutta secca i fichi. 

I dati ottenuti confermano l'alto rischio alimentare per prodotti provenienti da paesi terzi (Turchia, India, Iran, e 

altri) dove non sempre le condizioni di stoccaggio e trasporto delle derrate alimentari possono garantire la 

corretta conservabilità e salubrità dei prodotti. 

CONCLUSIONI 

Il consumo di prodotti da agricoltura biologica è in espansione ed è maggiore da parte di persone che hanno 

scelto un’alimentazione prevalentemente vegetariana. Il consumo maggiore di prodotti biologici viene registrato 

nel settore ortofrutticolo, con il 40% delle preferenze sul totale dei consumatori, seguito da pasta e riso con l’8 ed 

il 2-3% dei prodotti confezionati. I prodotti considerati nel presente lavoro hanno una larga diffusione tra i 

consumatori di prodotti biologici. 

I risultati ottenuti dalla indagine effettuata in questo biennio sono stati soddisfacenti e ci hanno permesso di 

ricavare le seguenti informazioni : 

• Sono necessari controlli mirati sia allo stoccaggio presso i centri di produzione (per cereali di 

produzione locale) sia nella grossa distribuzione (ipermercati); 

• Un più attento controllo deve essere effettuata su matrici a particolare rischio quali semi oleaginosi 

(mandorle, pistacchi, arachidi e noci), spezie (peperoncino e polveri derivate), caffè; e in particolare quelle 

provenienti da paesi terzi dove non sempre le condizioni di trasporto e stoccaggio garantiscono la salubrità 

dei prodotti; 

• Devono essere in oltre monitorati i prodotti locali insilati quali grano, riso, sfarinati. 

Infine va ricordato che gli alimenti che sono risultati maggiormente a rischio di aflatossine sono arachidi e 

derivati, mais e derivati, noci brasiliane, mandorle, fichi secchi, alcune spezie (peperoncino); ma che una cattiva 

conservazione può far comparire le aflatossine anche in prodotti non considerati a rischio. 

Bibliografia 

- Nasir M.S., Jolley M.E., “Development of a fluorescence polarization assay for the determination of 

aflatoxins in grains”. J. Agric. Food Chem. 50(11):3116-21 (2002). 

- Schatzki T.F., Haddon W.F., “Rapid, non-destructive selection of peanuts for high aflatoxin content by 

soaking and tandem mass spectrometry”. J. Agric. Food Chem. 50(10):3062-9 (2002). 


- Sobolev V.S., Dorner J.W., “Cleanup procedure for determination of aflatoxins in major agricultural 

commodities by liquid chromatography”. J. AOAC Int. 85(3):642-5 (2002). 

- Regolamento CE 1525/98 

- Circolare n°10 del 09/06/99 Gazzetta Ufficiale della Repubblica Italiana 

- Barbieri G., Bergamini C., Ori E, Resca P. Journal of Food Science 1313 -1331(1994) 

- Finoli C., Ferrari M. Industrie Alimentari, 732-736 (1994) 

- Brera C., Miraglia M., Colatosti M.: Microchemical Journal, 59 (1), 45-49 (1998) 

- Brera C., Miraglia M. Microchemical Journal, 54 (4), 465 -471 (1996) 

- R.C. Garden, M.M. Wattman, P.J.L. Taylor and M.W Stow Journal of Chromatography, 648, 485-490 

(1993) 

- Brera C. Rapporti ISTISAN 34, 203-214 (1996) 


116

22 

PEPTIDATI DI RAME: PRODOTTI INNOVATIVI 

A BASSO DOSAGGIO A BASE DI RAME, CHELATO AD AMINO ACIDI E PEPTIDI 

Paolo Maini 

U.R. GRIFA no. 42 - c/o SICIT 2000 SpA, Chiampo VI 

Il rame, insieme allo zolfo, è forse il più indispensabile dei prodotti utilizzati per la difesa delle colture 

nell'Agricoltura Biologica (AB). Vite, patata, pomodoro, olivo e molte frutticole non si potrebbero assolutamente 

produrre senza rame in AB. 

A livello europeo, i Paesi del Nord vorrebbero eliminare del tutto il rame dalla lista dei prodotti ammessi, a causa 

dell'inquinamento delle falde acquifere in quei suoli, principalmente acidi. Nel Sud Europa la prevalenza di suoli 

alcalini evita la percolazione e quindi il problema è meno sentito. E’ vero però che i Paesi del Nord sono i 

consumatori di una buona parte delle nostre produzioni biologiche e quindi le loro richieste devono essere tenute 

in debita considerazione. A livello normativo (Reg. CEE 2092/91) l’utilizzo del rame era stato concesso fino al 

31 marzo 2002, e ciò avrebbe significato la morte dell'AB, se non fossero intervenuti gli organismi dei 

produttori biologici con proposte di riduzione dell’uso al minimo. Fortunatamente, è stata accettata la proposta di 

"…una riduzione graduale dell’uso del rame, con un limite massimo di 8 kg di rame metallico per ettaro e per 

anno. Dopo 4 anni, il rame dovrà essere ulteriormente ridotto e successive riduzioni verranno decise in base al 

progresso della ricerca sulle possibili alternative. La quantità di rame ad ettaro e per anno andrà calcolata come 

media quinquennale, in modo da poter fronteggiare annate climaticamente avverse con quantità leggermente 

maggiori di rame, per poi recuperare nelle annate meno difficili." 

Ed è proprio sulla linea delle suddette normative che si posizionano i nuovi ed innovativi formulati a base di 

rame complessato ad amino acidi e peptidi (PEPTIDATI o Polipeptidati di rame), capaci di ridurre fortemente i 

dosaggi del rame ad ettaro. 

Peptiram 5 e Peptiram 7, peptidati dim rame chelato ad amino acidi e peptidi, rispettivamente a base di solfato e 

di idrossido di rame e registrati dalla SICIT 200 SpA nell'aprile del 2002, si differenziano nettamente da tutti i 

prodotti fungicidi convenzionali a base di rame. Essi possono rappresentare, pertanto, una nuova e valida 

alternativa nel controllo delle malattie fungine e batteriche delle colture agrarie, particolarmente nell'AB, 

tenendo in conto anche il fatto che il rame, pur essendo utilizzato da più di cento anni, non ha mai prodotto 

fenomeni di resistenza. 

I due nuovi Peptidati di rame sono caratterizzati da una speciale struttura basata su complessi del rame con 

amino acidi e peptidi (Figura 1). Il metallo, così legato, non segue più la normale via di penetrazione nelle 

cellule vegetali con meccanismo “cationico”, ma penetra con gli stessi meccanismi delle sostanze organiche. La 

forte riduzione dei dosaggi/ha di rame che ne deriva, può arrivare fino ad un quinto o ad un decimo, 

rispettivamente con Peptiram 5 e Peptiram 7. 

Figura 1: Esempi di chelati tra Rame ed amino acidi. A sinistra: 1 atomo di Cu, 2 moli di Glicina e 1 mole di 

H2O; a destra:1 atomo di Cu , 1 mole di Istidina e 1 mole di Asparagina . 

Forse proprio per questo meccanismo di facile penetrazione è da notare l'efficacia mostrata verso malattie 

fungine solitamente non controllate dal Cu, quali certe ruggini. Mentre in altre colture (ad es:. olivo, 

cucurbitacee) prevale l'azione stimolante degli amino acidi con notevoli rigogli vegetativi e produttivi, in alcune 

colture (ad es.: vite), a causa della rapida assimilazione, si possono verificare fenomeni fitotossici, in funzione 

della varietà, dello stadio fisiologico o delle condizioni ambientali. L'importanza dell'impiego del rame nella 

vite, ha portato a saggiare la sensibilità varietale su 16 cv., con buoni risultati, escluso il moscato. 


117

La necessità di dover ridurre i dosaggi di rame per ettaro, ha spinto vari Organismi regionali o affini, che si 

occupano di AB, a fare numerose prove con formulazioni o tecniche nuove. Negli ultimi due-tre anni anche i 

Peptiram sono stati inseriti in tali programmi e generalmente i risultati sono stati tra i più soddisfacenti. 

Nel corso di questa ultima annata, dopo la registrazione dei due prodotti, sono state impostate anche numerose 

prove dimostrative su varie colture. 

L'impiego dei due Peptiram, inoltre, è del tutto soddisfacente anche sul piano igienico-tossicolgico, in quanto 

analisi dei residui di rame su vite e pomodoro hanno evidenziato valori non diversi dai testimoni (Figura 2). Ciò 

potrebbe essere particolarmente interessante per i produttori biologici di vini passiti, ove, in qualche caso, si 

sfiorano i livelli residuali massimi. 

Residui Cu , mg/kg 

3 

1,5 

0 

0,91 

2,16 

Controllo Peptiram 5 

6 kg/ha 

1,42 

Peptiram 7 

1,5 kg/ha 

Figura 2: Residui di Cu (mgKg -1 ) da prove ed analisi secondo le BPL. 

118 

UVA 

POMODORO 

0,72 

Controllo Peptiram 5 

300 g/100L 

0,82 0,84 

Peptiram 7 

100 g/100L 


23 

CHARACTERIZATION OF ORGANIC VIRGIN OLIVE OILS FROM CORATINA CULTIVAR 

E. Perri, N. Lombardo, I. Muzzalupo, E. Urso, M. 

Pellegrino 

Istituto Sperimentale per l’Olivicoltura 

C.da Li Rocchi, 87036 Rende (CS), Italy 

eperri@libero.it 

G. Sindona, C. Benincasa 

Dipartimento di Chimica 

Università della Calabria 

I-87030 Arcavacata di Rende (CS), Italy 

119 

C. Cavallo 

Regione Puglia 

I.P.A., Brindisi, Italy 

Introduction 

In spite of the growing interest in organically cultivated olive grove and organic olive oil in European and 

international markets, there are very few reports on the qualitative, nutritional and organoleptic characteristics of 

these products (Gutierrez et al., 1999; Perri et al., 1999; Perri et al., 2001; Perri et al., 2002). Recently, a 

research project dealing with the “Characterisation of olive oils from the South of Italy obtained by organic 

farming systems” has been co-funded by the EU and the Italian Ministry of Agriculture (B07 Project, 

Multiregional Operational Programme, 2 Measure). The principal aim of this research project was to classify and 

characterise the nutritional and organoleptic quality of organic olive oils and to compare the quality of organic 

olive oil of important Italian cultivars from selected producing areas to conventional and/or integrated olive oils 

from the same cultivars and areas. This paper presents some results of the second year of activity in Apulia of the 

above research project. 


Plant material. In 2000, triplicate samples of organically and integrated cultivated olive drupes of Coratina cv 

from two adjacent farms at Andria (Bari, Italy) were collected 10 days before the traditional start of harvest, in 

the middle of the harvest period and 10 days after the end of harvest time. To compare organically produced 

olive oils versus integrated olive oils the olives were handpicked from the same cultivars under comparable 

pedo-climatic, harvesting and productive conditions and the analytical and sensory data compared on the basis of 

the same stage of olive maturity monitored by Jaen index (Gutierrez et al., 1999). 



Analytical procedures. The measurements of free acidity, fatty acid methyl esters, peroxide index, specific 

extinction coefficients K232 and K270, ∆k, and panel test have been determined according to the European Official 

Methods of Analysis (EC Regulation N. 2568/91 July, 1991). Total phenols were determined by colorimetry 

using the Folin-Ciocalteu reagent using caffeic acid as external standard. Oleuropein (olp) was determined in 

virgin olive oil extracts by tandem mass spectrometry with Atmospheric Pressure Chemical Ionization (APCI- 

MS/MS) under Selected Reaction Monitoring (SRM) condition (Perri et al., 1999b). 


The analytical data arising from the second year of sampling confirm the preliminary results published on the 

first paper (Perri et al., 2002): i) olive oil from Coratina cv. is characterised by high level of total phenols and 

high percentages of oleic acid in a wide range of stages of maturity of the drupes; ii) all the examined olive oil 

samples were ascribable to the extra virgin olive oil grade on the basis of analytical data relating to free acidity, 

peroxide number, specific extinction coefficients K232 and K270 , ∆k, fatty acid methyl esters, sterol and 

organoleptic score (panel test); iii) ecologically grown olives can produce high-quality oil thanks to the low 

Bactrocera oleae infestations; iv) as shown by the variance analysis (table 1), analytical and sensory data 

relating to organic and integrated olive oils are very similar. In fact, the only statistical significant difference 

observed between organic and integrated olive oils corresponding to the means of the sensory score of integrated 

olive oil (table 1) may be considered negligible from the quality point of view. 


The Italian Ministry of Agriculture and European Union (Multiregional Operational Programme, Measure 2, B07 

Project) supported this research work. We thank Dr. Nino Paparella, Dr. Nicola Panaro (C.I.Bi., Bari) and Drs. 

Edoardo and Giancarlo Ceci Ginistrelli for olive sampling, data collection on farming systems and olive grove 

management. We thank also Prof. Angelo Putignano and Prof. Francesco Prudentino (I.T.A.S. of Ostuni-BR) for 

olive oil extraction. 

Literature cited 

Gutierrez R. F., Arnaud T., Albi M., Influence of ecological cultivation on virgin olive oil quality, JAOCS, 1999, 

76:617-621. 


Perri E., Palopoli A., Pellegrino M., Sirianni R., Miele D., 1999a, La caratterizzazione degli oli di oliva calabresi 

ottenuti da agricoltura biologica, Proceeding of Seminario-Laboratorio “Metodi e sistemi innovativi 

dell’olivicoltura biologica e sostenibile: stato della ricerca e della sperimentazione”, Rende, april 14-16, pp147- 

153. 

Perri E, Raffaelli A., Sindona, 1999b, G., Quantitation of oleuropein in virgin olive oil by ionspray mass 

spectrometry-selected reaction monitoring, J. Agric. Food Chem., 47(10), 4156-4160. 

Perri E., Rizzuti B., Pellegrino M., Salvo F., Spartà G., 2001, Valutazione organolettica degli oli di oliva siciliani 

campionati nell’ambito del progetto POM B07 ”Caratterizzazione degli oli di oliva meridionali da agricoltura 

biologica” nell’annata 1999-2000, in Corso per tecnici degustatori d’olio extravergine e vergine d’oliva, 

Regione Siciliana, Alcamo. 

Perri E., Rizzuti B., Pellegrino M., Paparella N., Panaro N., Cavallo C., 2002, Characterisation of italian virgin 

olive oils from organic farming systems, Atti “4th Intenational Symposium on olive growing”, Valenzano (BA), 

25-30 settembre - Acta Horticulturae (in corso di stampa). 

Perri E., Lombardo N., Parlati M.V., Fodale A., Mulè R., Pellegrino M., Salvo F., Spartà G., 2002, 

Caratterizzazione di oli di oliva da agricoltura biologica provenienti dalla Sicilia, Atti del Convegno 

Iternazionale di Olivicoltura, Spoleto, 22-23 aprile 2002. 

Table 1. Mean values of the main quality parameters of olive oil of Coratina cv. from Andria (BA) and 

significant differences in the means of the same treatment and between treatments. 

Ripeness index 

Parameter Culture 2.2 3.2 Mean (a) 

Acidity Organic 0.28 n.s. 0.23 n.s. 0.26 

Integrated 0.28 n.s. 0.28 n.s. 0.28 

n.s. n.s. n.s. 

Peroxide Organic 2.93 n.s. 3.20 n.s. 3.07 

Integrated 2.60 b 3.27 a 2.93 


Total phenols Organic 485.80 n.s. 418.13 n.s. 451.97 



Organoleptic score Organic 7.00 n.s. 7.53 n.s. 7.27 


* n.s. n.s. 

Oleic acid Organic 76.73 n.s. 74.86 n.s. 75.79 



Oleuropein (mg/kg) Organic 0.126 A 0.090 B 0.108 

(a) Overall means. Differences within the same row (Tukey test): n.s.=not significant; different letters 

indicate a significant differences at P 0.05 (Tukey test). Differences within the same column (Tukey test): 

n.s.=not significant; * P

24 

CHARACTERIZATION OF ORGANIC VIRGIN OLIVE OILS FROM OGLIAROLA SALENTINA 

CULTIVAR 

E. Perri, N. Lombardo, I. Muzzalupo, B. 

Rizzuti, M. Pellegrino 

Istituto Sperimentale per l’Olivicoltura 

C.da Li Rocchi, 87036 Rende (CS), Italy 

eperri@libero.it 

G. Sindona, C. Benincasa 

Dipartimento di Chimica 

Università della Calabria 

I-87030 Arcavacata di Rende (CS), 

Italy 

121 

C. Cavallo 

Regione Puglia 

I.P.A., Brindisi, Italy 

Introduction 

The organic extra-virgin olive oil would be able to have high nutritional and sensory quality as necessary 

requirement to become part of those Italian products that are able to survive in the national and international 

market. However, there are very few reports on the qualitative, nutritional and organoleptic characteristics of 

these products (Gutierrez et al., 1999; Perri et al., 1999a; Perri et al., 2001; Perri et al., 2002). Recently, a 

research project dealing with the “Characterisation of olive oils from the South of Italy obtained by organic 

farming systems” has been co-funded by the EU and the Italian Ministry of Agriculture (B07 Project, 

Multiregional Operational Programme, 2 Measure). The principal aim of this research project was to classify and 

characterise the nutritional and organoleptic quality of organic olive oils and to compare the quality of organic 

olive oil of important Italian cultivars from selected producing areas to conventional and/or integrated olive oils 

from the same cultivars and areas. This paper presents some results of the second year of activity in Apulia of the 

above research project. 


Plant material. In 2000/2001 harvest, triplicate samples of organically and conventional cultivated olive drupes 

of Ogliarola salentina cv. from two adjacent farms at Villa castelli (Brindisi, Italy) were collected 10 days before 

the traditional start of harvest, in the middle of the harvest period and 10 days after the end of harvest time. To 

compare organically produced olive oils versus conventional olive oils the olives were handpicked from the 

same cultivars under comparable pedo-climatic, harvesting and productive conditions and the analytical and 

sensory data compared on the basis of the same stage of olive maturity monitored by Jaen index (Gutierrez et al., 

1999). 



Analytical procedures. The measurements of free acidity, fatty acid methyl esters, peroxide index, specific 

extinction coefficients K232 and K270, ∆k, and panel test have been determined according to the European Official 

Methods of Analysis (EC Regulation N. 2568/91 July, 1991). Total phenols were determined by colorimetry 

using the Folin-Ciocalteu reagent using caffeic acid as external standard. Oleuropein (olp) was determined in 

virgin olive oil extracts by tandem mass spectrometry with Atmospheric Pressure Chemical Ionization (APCI- 

MS/MS) under Selected Reaction Monitoring (SRM) condition (Perri et al., 1999b). 


According with the results of the first year of observation (Perri et al., 2002), from the data of quality parameters 

of table 1, it turns out that the olive oil from Ogliarola salentina cv. was characterised by medium to low level of 

total phenols and low percentages of oleic acid. Moreover, only the olive oils produced in the first period of 

harvest were ascribable without any tolerance to the extra virgin olive oil grade on the basis of organoleptic 

score (panel test). This is probably due to an increase of the percentages of Bactrocera oleae infestations 

observed in the second harvest period. As shown by the variance analysis (table 1), analytical and sensory data 

relating to organic and conventional olive oils are very similar probably because of similar percentages of olive 

fly infestation due to ineffective pesticide treatments. Finally, an increase of olp content in olive oil versus Jaen 

index was also observed. 


The Italian Ministry of Agriculture and European Union (Multiregional Operational Programme, Measure 2, B07 

Project) supported this research work. We thank Mr. Lorenzo Elia and Amalio Cassese for olive sampling, data 

collection on farming systems and olive grove management. We thank also Prof. Angelo Putignano and Prof. 

Francesco Prudentino (I.T.A.S. of Ostuni-BR) for olive oil extraction. 


Literature cited 

Gutierrez R. F., Arnaud T., Albi M., Influence of ecological cultivation on virgin olive oil quality, JAOCS, 1999, 

76:617-621. 

Perri E., Palopoli A., Pellegrino M., Sirianni R., Miele D., 1999a, La caratterizzazione degli oli di oliva calabresi 

ottenuti da agricoltura biologica, Proceedings of Seminario-Laboratorio “Metodi e sistemi innovativi 

dell’olivicoltura biologica e sostenibile: stato della ricerca e della sperimentazione”, Rende, april 14-16, pp147- 

153. 

Perri E, Raffaelli A., Sindona, 1999b, G., Quantitation of oleuropein in virgin olive oil by ionspray mass 

spectrometry-selected reaction monitoring, J. Agric. Food Chem., 47(10), 4156 -4160. 

Perri E., Rizzuti B., Pellegrino M., Salvo F., Spartà G., 2001, Valutazione organolettica degli oli di oliva siciliani 

campionati nell’ambito del progetto POM B07 ”Caratterizzazione degli oli di oliva meridionali da agricoltura 

biologica” nell’annata 1999-2000, in Corso per tecnici degustatori d’olio extravergine e vergine d’oliva, 

Regione Siciliana, Alcamo. 

Perri E., Rizzuti B., Pellegrino M., Paparella N., Panaro N., Cavallo C., 2002, Characterisation of italian virgin 

olive oils from organic farming systems, Atti “4th Intenational Symposium on olive growing”, Valenzano (BA), 

25-30 settembre - Acta Horticulturae (in corso di stampa). 

Perri E., Lombardo N., Parlati M.V., Fodale A., Mulè R., Pellegrino M., Salvo F., Spartà G., 2002, 

Caratterizzazione di oli di oliva da agricoltura biologica provenienti dalla Sicilia, Atti del Convegno 

Iternazionale di Olivicoltura, Spoleto, 22-23 aprile 2002. 

Table 1. Mean values of the main quality parameters of olive oil of Ogliarola salentina cv. from Villa Castelli 

(Brindisi, Italy) and significant differences in the means of the same treatment and between treatments. 

Ripeness index 

Parameter Culture 2.0 4.1 Mean (a) 

Acidity Organic 0.42 n.s. 0.37 n.s. 0.40 

(as % of oleic acid) Conventional 0.28 n.s 0.33 n.s. 0.30 


Peroxide (mg O2/kg) Organic 5.40 n.s. 5.80 n.s. 5.60 

Conventional 5.07 n.s. 5.40 n.s. 5.23 


Total phenols Organic 225.63 n.s. 180.13 n.s. 202.88 

(mg/kg) Conventional 266.23 A 177.10 B 221.67 


Organoleptic score Organic 7.00 A 5.53 B 6.27 

Conventional 6.66 a 5.53 b 6.00 


Oleic acid (%) Organic 66.22 n.s. 66.21 n.s. 66.22 

Conventional 65.15 n.s. 68.74 n.s. 66.94 


Oleuropein (mg/kg) Organic 0.073 A 0.120 B 0.096 

(a) Overall means. 

Differences within the same row (Tukey test): n.s.=not significant; means in row followed by the same letter are 

not significantly different at P 0.01 (capital letters) or P 0.05. Differences within the same column (Tukey test): 

n.s.=not significant, * P

25 

EFFICACY EVALUATION OF BIOLOGICAL CONTROL AGENTS AGAINST 

PLASMOPARA VITICOLA 

Ilaria Pertot, Federica De Luca, Antonella Vecchione, Luca Zulini 

Istituto Agrario di San Michele all’Adige, via E. Mach 1, S. Michele all’Adige (TN) 

ilaria.pertot@ismaa.it 

Keywords 

downy mildew, Plasmopara viticola, biological control agents, organic agriculture. 

Introduction 

Downy mildew causal agent, Plasmopara viticola (Berk. et Curt.) Berl. et De Toni, is one of the most important 

grapevine pathogens, causing great losses if no protective treatments are applied (Kortekamp, 1997). 

Nowadays consumer and farmer are much more concerned about food, health and environment safety (Butt, 

2001). The conditions for using copper will be restricted by European Union by fixing a ceiling on use expressed 

in terms of kilograms of copper per hectare per year in organic agriculture. Antagonist microorganisms could be 

a possible alternative to copper in downy mildew controlling. Epidemiological characteristics of downy mildew 

probably would not allow a complete field control based on a single biocontrol agent (BCA). A possible solution 

for improving efficacy of BCA could be the integration of several micro-organisms with different action 

mechanisms on specific stages of disease: i) during oospore overwintering and germination, for primary 

inoculum reduction ii) and during sporangia germination for protection against secondary infections. 


Micro-organisms were isolated from grapevine leaf material and rhizosphere of 18 different vineyards located in 

the north –east of Italy. Vineyards were abandoned and chemically untreated for at least three years. Microorganisms 

were isolated on potato dextrose agar, malt agar and nutrient sucrose agar and grown at 20°C. Several 

isolation methods were used: inocula were based on leaf washing water, necrotic leaf portions grounded in sterile 

water with mortar and pestle, necrotic leaf pieces, infected leaf dish maintained in contact with naturally 

degraded leaf material and root surface material. For each kind of material used, all morphologically different 

colonies of fungi and bacteria were collected. A sample of 46 isolates was evaluated for sporangia germination 

and infection inhibition activity. A sample of 33 isolates was evaluated for overwintering oospores inhibition 

activity. 

The evaluation methods used were: 

i) oospore germination inhibition, based on a modified floating leaf disk method (Hill); 

ii) inhibition of sporangia germination, based on reduction percentage of sporangia germination in a 1:1 

water solution of the isolate culture broth and P. viticola sporangia; 

iii) infection inhibition, based on the reduction of the number of sporangiophore developed on isolate 

culture broth treated leaves. 


Several micro-organisms have induced a partial inhibition of sporangia germination (tab. 1), but the complete 

control of infection on leaf can be achieved with only few of them. 


123

Tab. 1 - Biological Control Agents (BCA) efficacy on sporangia germination and 

infection process inhibition. 

BCA 

% germinated 

sporangia 

124 

Inhibition of infection 

process 

Level of 

inhibition 

1 3 yes C 

2 2 no A 

3 0 no B 

4 3 no A 

5 4 no A 

6 0 no B 

7 3 no A 

8 0 partial B 

9 2 yes B 

10 4 yes A 

11 0 no B 

12 0 no B 

13 untreated no - 

14 1 no A 

15 0 no B 

16 0 no B 

17 1 yes D 

18 1 no A 

A) No sporangia germination inhibition as well as infection process. 

B) Partial sporangia germination inhibition or infection process. 

C) Inhibition of infection process, but no inhibition of sporangia germination. 

D) Sporangia germination inhibition as well as infection process. 

Twenty micro-organisms have given a good control on conidiophore development (fig.1) with less than thirty 

conidiophore/cm2 developed. Best results have been found on oospore germination: 42% of the tested 

microorganisms completely inhibited of oospore germination (fig. 2), while the remaining 58% showed some 

degrees of efficacy on the oospore germination inhibition and on the time necessary for oospore germination in 

optimal conditions. 


Fig. 1 – Effects of Biological Control Agents (BCA) on sporangiophore development of P. viticola (in vitro leaf 

infections). 

Untreated 

Isolated 

18 

29 

22 

13 

15 

28 

20 

27 

1 

24 

5 

16 

19 

8 

14 

21 

25 

10 

3 

6 

26 

23 

17 

12 

11 

9 

7 

4 

2 

a 

ab 

bc 

abc 

0 10 20 30 40 50 60 70 80 90 100 

N° Sporangiophore/cm 2 


bcd 

125 

cde 

def 

ef 

efg 

fg 

g

Fig. 2 – Efficacy evaluation of BCA on overwintering oospore germination. 

BCA Efficacy on overwintering 

oospore germination. 

Not sporulated 

42% 

Sporulated 

58% 

N° of sporulated disk 

N° days for oospore 

germination 

16 

14 

12 

10 

8 

6 

4 

2 

0 

7 

6 

5 

4 

3 

2 

1 

0 

untreated 

112 

A strong reduction of the overwintering inoculum could be useful in downy mildew control, in fact new 

epidemiological studies have demonstrated that primary infections play an important role in the disease 

development (Pertot et al., 2002). If biocontrol activity of the studied micro-organisms is confirmed in field 

trials, the combined effect of several BCAs on the different biological stages of the pathogen will be evaluated. 

In such way BCAs could help in reducing copper quantity in organic viticulture. 


This work was funded by the Fund for Research of the Autonomous Province of Trento, Italy, Research project 

AGRIBIO. 

References 

Kortekamp A. (1997) Epicoccum nigrum Link: A biological control agent of Plasmopara viticola (Berk. et Curt.) 

Berl. et De Toni. Vitis 36 (4), 215-216. 

Butt T. M., Jackson C., Magan N. (2001) Introduction- Fungal Biological Control Agents: Progress, Problems 

and Potential. In: Fungi as biocontrol Agents. Progress, Problems and Potential. CABI Publishing, Wallingford, 

UK, 1-8. 

Hill G. () IOBC Bulletin. 

Pertot I., Gobbin D., Gessler C. (2002) Epidemiology of Plasmopara viticola: field distribution of primary and 

secondary infections in the early stage of the season and microclimate impact on infection severity. In: 

Proceedings of the 4th International Workshop on Powdery and Downy Mildew in Grapevine. Napa, California, 

U.S.A., September 30-October 04, 2002. 

126 

BCA which have partially inhibited the 

overwintering oospore germination. 

98A 

119A 

35 

22A 

112 

86 

2A 

2A 

69 

13 

37 

93 

10A 

34 

99 

Isolate 

44 

77F 

120F 

13 

37 

49A 

59A 

69 

93 

10A 

untreated 

Time for oospore germination in samples treated with 

BCA which have which have partially inhibited the 

overwintering oospore germination. 

Isolate 

98A 

119A 

22A 

99 

44 

77F 

120F 

59A 

35 

86 

34 

49A 


26 

IL CONTROLLO DELLE OPERAZIONI DI CAMPO COME BASE DELLA RINTRACCIABILITÀ A 

GARANZIA DEL CONSUMATORE E DELL’AMBIENTE 

Alessandro Pulga (AIAB – ICEA – Bologna) 

Ivano Valmori (Image Line – Banca dati Biolgest – Faenza) 

Dall’aprile del 2002 è stato creato un servizio su internet destinato alla gestione dei dati relativi alle operazioni 

colturali svolte presso le aziende agricole in coltivazione biologica. 

L’iniziativa ha avuto come obiettivo quello di raccogliere le informazioni relativamente a: 

- registrazione di tutti gli interventi di difesa fitosanitaria e controllo delle avversità (svolta con prodotti 

fitosanitari, biologici e naturali, ausiliari e sistemi basati su trappole e feromoni); 

- registrazione delle fertilizzazioni minerali ed organiche; 

- registrazione di tutte le operazioni di campo (lavorazioni, sfalci, etc.); 

- registrazione delle operazioni colturali (potature, diradamenti, raccolta, etc.); 

- registrazione degli eventi atmosferici; 

- registrazione dei conferimenti; 

- gestione dei magazzini aziendali; 

- tenuta del quaderno di campagna in base alla normativa 290/01 (registro dei trattamenti effettuati). 

Il tutto viene reso fruibile direttamente su Internet, sul sito www.quadernodicampagna.it, permettendo a tecnici 

ed agricoltori di interagire con il sistema. Gli agricoltori, di fatto unici depositari delle informazioni reali sul 

ciclo produttivo della merce, sono incentivati ad utilizzare il sistema in quanto ricevono dallo stesso una serie di 

agevolazioni operative quali: 

- controllo incrociato delle registrazioni dei prodotti utilizzati in base alle banche dati dei fitofarmaci; 

- verifica del rispetto degli intervalli di sicurezza; 

- verifica delle giacenze e delle scorte di magazzino; 

- simulazione delle operazioni colturali al fine di verificarne la correttezza. 

I vantaggi del progetto. 

Per i tecnici: 

- erogare un servizio tecnico direttamente dalla propria sede (ricetta on-line); 

- tenere traccia dei consigli forniti per ogni appezzamento di ogni agricoltore; 

- verificare la correttezza delle operazioni svolte. 

Per gli agricoltori: 

- massima semplicità di utilizzo del sistema; 

- possibilità di assolvere agli obblighi imposti dalla normativa vigente in materia di agricoltura biologica 

(registrazioni obbligatorie previste dal Reg. CEE 2092/91 e D.lgs. 220/95); 

- fornire tutti i dati utili richiesti dai clienti (in particolare dalla GDO); 

- nessun software da installare e aggiornare, ma collegamento al servizio tramite area riservata attraverso 

internet, anche con un semplice modem standard; 

- garanzia del salvataggio dei dati (backup); 

- disponibilità della versione sempre più efficiente e con banche dati aggiornate settimanalmente. 

Per gli Organismi di Controllo e le autorità pubbliche di sorveglianza: 

- possibilità di acquisire tutte le informazioni – utili ai fini del controllo e della certificazione – in formato 

elettronico; 

- possibilità di dialogo e di scarico dei dati nei propri sistemi informatici; 

- i dati in formato elettronico sono più facilmente fruibili e permettono controlli più veloci ed efficaci. 

Per i consumatori ed il mercato: 

- massima trasparenza nel ciclo produttivo e garanzia di poter risalire all’origine del prodotto ed entrare 

nel dettaglio di tutti gli interventi effettuati dal campo alla tavola; 

- possibilità di sapere cosa si consuma. 

Questo sistema costituisce sicuramente un primo importante strumento per creare la base dati fondamentale per 

sviluppare sistemi di rintracciabilità, dalle fasi a monte della filiera produttiva fino al consumatore finale. 

Questo sistema, opportunamente implementato e validato da un Ente di certificazione di parte terza, può 

diventare strumento utile a tutte le organizzazioni interessate, al fine dell’ottenimento di certificazioni volontarie 

di prodotto e della certificazione di rintracciabilità di filiera in conformità alla norma UNI 10939. 


127

27 

ECO-TOXICITY OF THE ENTOMOPATHOGENIC BACTERIUM-NEMATODE SYMBIOTIC 

COMPLEX TOWARD NON-TARGET ORGANISMS 

M. Ricci, M. Colli, R. Barcarotti and A. Ragni 


Tel: ++39 – 075 - 895091 Fax: ++39 – 075 – 888 776 E-mail: mricci.bt@parco3a.org 

Introduction 

In agriculture, the use of bio-control agents is growing; this is due, in particular, to their ability to avoid pest’s 

resistance and to their friendly environmental impact. 

A mini-review of the entomopathogenic bacterium-nematode symbiotic complex toward non-target organisms is 

presented; bibliographic data and original data coming from research are shown. In particular it is mentioned a 

work which first demonstrated specificity of the entomopathogenic bacterium-nematode symbiotic complex 

toward target organisms (insects). 

The present work concerns three sections related to the eco-toxicity of entomopatogenic nematodes: a) Short 

review; b) Demonstration of entomopathogenic nematodes host specificity; c) Laboratory results toward nontarget 

hosts: Earthwoms, Scorpions and Millipeds. 

a) Short review 

Poinar (1990) reported that although Steinernematids and Heterorhabditids are considered entomopathogenic 

nematodes because all natural infections have involved insects, they do have the ability to enter and kill noninsect 

invertebrates, at least in the laboratory (Wayne et al., 1987). Steps of their infection process are normally 

completed in most insects but various obstacles to their completion occur with many non-insect invertebrates 

(Wayne et al., 1987). The effect of these nematodes on vertebrates is of a great concern. Off all the vertebrates 

(among the following classes: Pisces, Reptilia, Amphibia, Aves and Mammalia) thus far challenged with 

infective stage of Steinernematids and Heterorhabditids, only young tadpoles of frogs and toads were vulnerable. 

But the reason was given to the fact that when penetrating into the host the nematodes were carrying foreign 

bacteria. 

b) Demonstration of entomopathogenic nematodes host specificity ( Ricci et al., 1994) 

Introduction 

Entomopathogenic nematode infective juveniles (IJs) locate and parasitise potential hosts. The success of this 

process depends with suitability of the host. Ricci et al. (1994) and Lewis et al. (1996), demonstrated that a 

nematode's subsequent degree of attraction to CO2, stimulated by contact with the cuticle of an arthropod, 

should signify that the exposed IJs recognize it as a potential host. The level of stimulation caused to S. 

carpocapsae, a nematode that forages primarily by ambushing passing hosts, by 12 potential hosts (3 

Lepidoptera, 4 Coleoptera, 1 Orthoptera, 1 Blattodea, 1 Diptera and 2 non-insect arthropods) was compared. To 

determine whether arthropods that stimulate a high level of attraction of IJs are suitable hosts, three parameters 

were tested: nematode induced mortality, nematode invasion rate and reproductive potential. 


Behavioral Recognition Assay: nematodes were exposed for a certain time to the cuticle of different insects or 

non-insects or inert materials, then exposed to chemical volatile cues emitted by the insects Galleria mellonella 

in an apparatus called Agar Plate Arena (see Figure 1). After a certain time the nematodes that accumulated in 

the proximity of the cue emissions, were collected and counted. 

Nematode Infectivity Assay: host mortality was recorded after 2 days in Petri dishes at the concentrations of 200 

and 500 nematodes per host. 

Nematode Invasion Assay: Nematodes that penetrated the host during the Infectivity assay were counted. 

Reproductive Potential Assay: pre-weighed hosts were exposed to 500 IJs. After 2 days the cadavers were 

transferred on a nematode collecting trap. Every 2 days, the emerged IJs were collected until the exploitation of 

all the host tissues was completed. 


128

Results and Discussion 

S. carpocapsae was differentially stimulated to be attracted to G. mellonella volatiles by the contact of the 

cuticle of different arthropods. Agrotis ipsilon pupa, Leptinotarsa decemlineata, Isopoda sp., Blattella 

germanica, Musca domestica and Diplopoda sp. were not significantly different from the controls. A. ipsilon 

larva, Tenebrio molitor, Acheta domesticus, G. mellonella, Diabrotica virgifera virgifera and Popilia japonica 

induced a significantly higher "activation" to S. carpocapsae IJs compare to the controls. It seems that S. 

carpocapsae can discriminate even among different stage of the same species in fact the value of A. ipsilon larva 

is more than twice that of the pupa. 

The same hosts that induced the higher attraction to the volatiles were more susceptible to the nematodes. 

The nematodes, established (penetration rate and reproduction index) better in the group of arthropods that 

where better stimulated by G. mellonella volatiles. 

It has been demonstrated that, at least one entomopathogenic nematode, S. carpocapsae, could discriminate 

among suitable and not suitable hosts. 

Figure 1: Agar Plate Arena 

Glass plate 

G. mellonella larvae 

Pipette 

tip 

Chamber with gradient 

Nematodes 

Agar substrate 

Rubber 

c) Laboratory results toward non-target hosts: 

Application point of nematodes 

The non-insect organisms, earthworms, scorpions and millipedes have been challenged with different strains of 

entomopathogenic nematodes. 

Material and methods 

Hosts: the non-insect organisms, earthworms, scorpions and millipedes have been captured under stones, and 

stored at 12°C for 24 hours prior the experiment. 

Nematodes: were reared in Galleria mellonella last instar larvae by standard rearing procedures. 

For earthworms the following nematode strains were tested: Heterorhabditis bacteriophora NJ, Steinernema 

kraussei N0093 and S. feltiae UK. 

For Scorpions and Millipedes: H. bacteriophora NJ, S. kraussei N0093, S. sp. N0166, S. sp. N0167, S. sp. 

N0168, H. sp. N0169, H. sp. N0170. The infectivity of nematodes was tested in parallel on 100 mg larvae of 

Tenebrio molitor (10 larvae per box with 1500 nematodes). There were 3 repetitions per treatment. Incubation 

was done at 23°C for 5 days. Controls (only water) were always included. 

Tests with Earthworms: in 10x7x5 cm plastic boxes, 50 g of peat with 75% humidity, 5 earthworm were 

challenged with 3000 infective nematodes. There were 3 repetitions per treatment. Incubation was done at 23°C 

for 7 and 14 days. 

Tests with Millipedes: in 10x7x5 cm plastic boxes, 50 g of peat with 75% humidity, 3 millipedes were 

challenged with 1500 infective nematodes. There were 3 repetitions per treatment. Incubation was done at 23°C 

for 5 days. 

Tests with Scorpions: they were tested in Petri dishes with 3000 nematodes per individual for 5 days at 23°C. 

Results 

Mortality of Earthworms challenged with entomopathogenic nematodes (see Tab. 1) did not differed among 

treatments both at 7 (P=05957) and 14 days (P=0,7520). 

129 

Plexiglas lid 


Tab. 1 

Treatments Average Earthworm 

mortality at 7 days 

130 

Average Earthworm 

mortality at 14 days 

Steinernema feltiae UK 6,67 % 6,67 % 

S. kraussei N0093 20,00 % 20,00 % 

Heterorhabditis bacteriophora NJ 0,00 % 0,00 % 

Control 33,33 % 33,33 % 

Mortality of the Insect Tenebrio molitor and the non-insects Millipedes and Scorpions challenged with 

entomopathogenic nematodes (see Tab. 2) revealed as follows: the effect of nematodes is not different from the 

control in Millipedes (P=0,66) and in Scorpions (P=1) 

Tab. 2 

Treatments Tenebrio molitor 

larvae (insects) 

mortality 

Millipedes 

(non-insects) 

mortality 

Scorpions 

(non-insects) 

mortality 

S. kraussei N0093 73,33 % 0,00 % 0,00 % 

H. bacteriophora NJ 86,67 % 0,00 % 0,00 % 

S. sp. N0166 73,33 % 0,00% 0,00 % 

S. sp. N0167 93,33 % 0,00 % 0,00 % 

S. sp. N0168 40,00 % 0,00 % 0,00 % 

H. sp. N0169 6,67 % 11,11 % 0,00 % 

H. sp. N0170 96,67 % 11,11 % 0,00 % 

Control 0,00 % 11,11 % 0,00 % 

Conclusions and Discussion 

Unlike other microbial pesticides, such as Bacillus thuringiensis, viruses and fungi, entomopathogenic 

nematodes are exempt from registration in the USA and other countries.Currently, all available evidence 

(bibliography) indicates that beneficial nematodes and their associated bacteria have no negative impact on nontarget 

organisms in the agrarian environment (Akhurst 1990; Georgis et al., 1991). 

Entomopathogenic nematodes have the ability to discriminate among suitable and not suitable different potential 

hosts. 

Recent experiments (see above), demonstrated that while different strains of entomopathogenic nematodes were 

lethal to insect larvae (Tenebrio molitor), did not infect non-target organisms such as Heartworms, Scorpions and 

Millipedes. 

In conclusion entomopathogenic nematodes are safe to humans and to non-target organisms, non-polluting and 

thus environmentally safe and acceptable. 

Acknowledgment 

This research was partially founded by MURST grant Cluster C06-07 L. 488-92 

References: 

Akhurst, R.J. 1990. Safety to nontarget invertebrates of nematodes of economically important pests. In: Laird et 

al., pp. 233-240. 

Georgis, R., Kaya, H.K. and Gaugler, R. 1991. Effect of steinernematid and heterorhabditid nematodes 

(Rhabditida: Steinernematidae: Heterorhabditidae) on nontarget arthropods. Environ. Entomol. 20(3) : 815-822. 

Lewis E.E., Ricci M. and Gaugler R., 1996. Host recognition behaviour predicts host suitability in the 

entomopathogenic nematode Steinernema carpocapsae (Rhabditida: Steinernematidae) Parasitology (1996) 113- 

6, 573-581 

Poinar Jr, G.O., 1990. Taxonomy and Biology of Steinernematidae and Heterorhabditidae . In: (Eds. R. Gaugler 

and H.K. Kaya) Entomopathogenic nematodes in biological control, pp. 23-60. CRC Press, Boca Raton, U.S.A. 

Ricci M., Lewis E.E. and Gaugler R., 1994. Host discrimination by Steinernema carpocapsae (Weiser) All strain; 

at Debrecen, Hungary: Cost 819 Symposium & Workshop on Entomopathogenic Nematodes. In: C.T. Griffin, 

R.L. Gwynn & J.P. Masson (Eds.) COST 819, Biotechnology, Ecology and transmission strategies of 


entompathogenic nematodes. CSC-EC EAEC, Brussels, Luxembourg. p. 105 Smart Jr., G.C., 1995. 

Entomopathogenic Nematodes for the Biological control of Insects . Supplement to the J. of Nematology (27), 

529-534. 

Wayne, L.G., Brenner, D.J., Colwell, R.R., Grimont, P.A.D., Kandler, O., Krichevsky, M.I., Moore, L.H., 

Moore, W.E.C., Murray, R.G.E., Stackebrandt, E., Starr, M.P. and Truper, H.G., 1987. Report on the ad hoc 

committee on reconciliation of approaches to bacterial systematics . Int. J. Syst. Bacteriol., 37, 463. 


131

28 

PHOTORHABDUS AND XENORHABDUS: A NEW SOURCE OF USEFUL COMPOUNDS FOR 

BIOLOGICAL CONTROL 

A. Ragni and F. Valentini 


Tel: ++39 – 075 - 895091 Fax: ++39 – 075 – 888 776 E-mail: aragni.bt@parco3a.org 

IntroductionThe genera Photorhabdus and Xenorhabdus are symbiotic Enterobacteriaceae bacteria 

associated with entomopathogenic nematodes of the genera Heterorhabditis and Steinernema, respectively. The 

characteristics of both Photorhabdus and Xenorhabdus genera have been reviewed by Forst & Nealson (1996), 

and by Forst et al. (1997). 

Recently Bowen et al. (1998a; 1998b) and Blackburn et al. (1998) discovered and characterized an insecticidal 

toxin complex that it is secreted into the culture medium by Photorhabdus luminescens strain W-14. Except for 

this specific strain, the insecticidal capability of entomopathogenic soil nematode symbiotic bacteria has been 

reported only when carried into the insect by the nematodes or artificially injected into their haemocel. 

This work reports the isolation of a unique strain Photorhabdus sp., named XP01, selected among 12 

Photorhabdus and Xenorhabdus isolates, that displays an insecticidal activity when fed to the neonate larvae of 

several Lepidoptera pests.Materials and Methods. 

Bacteria isolation 

The bacteria strains tested were isolated as described by Akhurst (1980), from cadavers of Galleria mellonella 

infected with Heterorhabditis spp. or with Steinernema spp. strains belonging to the collection of 

entomopathogenic nematodes held at BioTecnologie S.r.l (BT) laboratories. A list of the isolated strains is 

shown on Table 1. Cultures were maintained by weekly subculturing on Nutrient Agar plates and incubated in 

the dark at 25°C. Inocula for liquid cultures were prepared as fermented broth aliquots containing glycerol at a 

final concentration of 15% (v/v) and stored at -80°C. 

Bacterial preparations 

Whole cultures were obtained by growing the strains in a Casein peptone plus yeast extract liquid medium in the 

dark at 25°C and shaking at 180 rpm , for a period of either 19 or 24 hours. Technical powder of XP01 was 

prepared by freeze-drying the pellet obtained by centrifugation the bacterial culture at 6,238 x g for 40 min. The 

pellet was washed three times in a neutral Ringer’s solution prior freezing. Freeze-drying resulted in cells dead 

since plating the resuspended technical powder gave no colony growing on Nutrient Agar. 

Insects rearing and bioassay techniques 

Colonies of Mamestra brassicae, Cydia molesta, Spodoptera littoralis, Helicoverpa armigera, Scotia segetum 

and Agrotis ipsilon are continuously reared at BT insectarium under controlled environmental conditions and fed 

with semi-synthetic diets. 

Insecticidal activity of bacterial suspensions was measured by using an overlay technique bioassay. Bacterial 

preparations at different serial dilutions were applied in 50 µl aliquots in the wells of a diet bioassay trays 

(surface area 175 mm 2 ) containing 1 g of an insect meridic diet. As control, the insect diet was treated with 

deionized sterile water. After drying, one unfed 1st instar larva (neonate) was placed in each well. Each 

treatment was applied to 32 insects. Mortality was scored after 5 days of incubation at 27°C. 

Results. 

Preliminary Screenings 

A total of 12 Photorhabdus and Xenorhabdus bacterial isolates were assayed for insecticidal activity against 

neonates of Mamestra brassicae and of Cydia molesta in a surface contamination assay. As shown in Figure 2, 

neonates of Cydia molesta were more susceptible than those of Mamestra brassicae when exposed to fermented 

broths of Photorhabdus and Xenorhabdus isolates. Treatments with XP01 and XP04 fermented broths resulted in 

100 % mortality of C. molesta neonates. In this species the mortality rate ranged from 25 to 41% for treatments 

with XP136, XP1007 and XP127. Low activity (from 0% to 19% mortality) against C. molesta neonates was 

recorded when treatments were done with the remaining bacterial strains. A 100% mortality of Mamestra 

brassicae neonates was observed when the insects were treated with XP01. A lower mortality equal to 74% was 

observed when XP04 was used. A residual activity (5% larval mortality) was recorded when treatment was done 

with strain XP10. 


132

After this preliminary screening and due to the high activity of XP01 against both larvae insect tested, this strain 

was selected for further characterization of its insecticidal activity. 

The XP01 activity was further tested on several other lepidopteran species. For convenience purposes only 

macrolepidopteran species were used in these assays. Results of this second screening are shown in Figure 3. 

The whole culture of XP01 showed the capability to kill all the five species treated, being most effective against 

M. brassicae, S. segetum and A. ipsilon. 

The insecticidal activity of freezed dried technical powder, made from the cell pellet of fermented broth, was 

tested against neonates of M. brassicae. As seen before with other bacterial preparations, the observed mortality 

of insects was proportional to the applied dose (Figure 4). 

Discussion. 

Photorhabdus and Xenorhabdus species are bacteria known to live in symbiotic association with 

entomopathogenic soil nematodes. Mechanisms involved in the pathogenicity of these bacteria resulting from 

their release by entomopathogenic nematodes in insects, have been extensively studied. A great deal of 

variability in the bacterial dose needed to kill different insects has been reported. The effect both depending on 

the bacterial strain used as well as the target insect studied. 

The present study describes the discovery of a newly characterize strain of Photorhabdus luminescens, 

denominated XP01, which is highly toxic when directly administered by food contamination assays to neonates 

larvae of several Lepidopteran pests including Mamestra brassicae and Cydia molesta. 

It was demonstrated that the toxic effect of XP01 is maintained even when a technical powder is used. Since 

technical powders do not contain alive cells, this result indicates that once the toxin has been synthesized alive 

bacterial cells are not further required for activity. This characteristic of XP01 insecticidal capability is similar to 

what has been described for Bacillus thuringiensis toxins (Schnepf et al., 1998). 

The discovery of the insecticidal activity of Photorhabdus luminescens strain XP01 indicates the existence of a 

presumably new family of entomotoxic proteins present in Photorhabdus and probably Xenorhabdus spp. These 

insecticidal toxins could be used as an active bio-pesticide ingredient in a similar manner to the delta-endotoxins 

of B. thuringiensis. This new source of natural toxins could help both the fight against the development of 

insects resistance as well as serve as sources of novel specific activities in combination with other known 

biopesticides.Acknowledgements 

We thank G. Flek for rearing insects for tests, L. Quattrocchi and R. Lorenzini for their assistance in 

microbiology and bioassay works. 

References 

Akhurst, R.J. 1980. Morphological and Functional Dimorphism in Xenorhabdus spp., of bacteria symbiotically 

associated with insect pathogenic nematodes Neoplectana and Heterorhabditis. J. Gen. Microbiol. 121: 303- 

309. 

Blackburn, M., E. Golubeva, D. Bowen, and. R.H. Ffrench-Constant. 1998. A novel insecticidal toxin from 

Photorhabdus luminescens, toxin complex a (Tca), and its histopathological effect on the midgut of Manduca 

sexta. Appl. Environ. Microbiol. 64: 3036-3041. 

Bowen, D.J., T.A. Rocheleau, M. Blackburn, O. Andreev, E. Golubeva, R. Bhartia, and R.H. Ffrench-Constant. 

1998a. Insecticidal toxin from the bacterium Photorhabdus luminescens. Science. 280: 2129-2132. 

Bowen, D.J., and J.C. Ensign. 1998b. Purification and characterization of a high-molecular-weight insecticidal 

protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens. Appl. Environ. 

Microbiol. 64: 3029-3035. 

Forst, S. and K. Nealson. 1996. Molecular biology of the symbiotic-pathogenic bacteria Xenorhabdus and 

Photorhabdus spp. Microbiol. Rev. 60: 21-43. 

Forst, S., B. Dowds, N.E. Boemare, and E. Stackebrandt. 1997. Xenorhabdus and Photorhabdus spp.: bugs that 

kill bugs. Annu. Rev. Microbiol. 51: 47-72. 

Schnepf, E., N. Crickmore, J. Van Rie, D. Lerecluse, J. Baum, J. Feitelson, D.R. Zeigler, and D.H. Dean. 1998. 

Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62: 775 -806. 


133

Photorhabdus strain XP01cells showing inclusion bodies. 

TABLE 1. Bacterial isolates tested 

Bacterial isolate Bacterial genus or species 

XP01 Photorhabdus luminescens 

XP1007 

XP127 

XP81 

XPHDO 

XPLon 

XPMib 

XP04 

XP05 Xenorhabdus sp. 

XP10 

XP136 

XP98 

FIG. 2. Mortality of neonates of two Lepidopteran pests treated with non-diluted fermented broth of several 

bacteria strains. Bacterial broths were normalized in terms of number of cells per ml before the use. Each bar 

represents the percent mortality obtained treating a total of 32 insects. 

Larval mortality (%) 

120 

100 

80 

60 

40 

20 

0 

XP01 XP04 XP05 XP10 XP1007 XP127 XP136 XP81 XP98 XPHDO XPLon XPMib 

Strain 

Mamestra brassicae Cydia molesta 


134

FIGURE 3. Insecticidal activity of fermented broth of XP01 strain against neonates of Mamestra brassicae, 

Spodoptera littoralis, Helicoverpa armigera, Scotia segetum and Agrotis ipsilon. n= 32 larvae for each treatment 

FIGURE 4. Insecticidal activity of fermented broth XP01 strain and of XP01 technical powder (resuspended to 

the original broth concentration of suspended solids) against neonates of Mamestra brassicae. Each point 

represents the percent mortality obtained treating a total of 96 insects in 3 replicate bioassays with 32 insect 

each. 


100 

80 

60 

40 

20 

0 


100 

80 

60 

40 

20 

0 

1:1 1:4 1:8 1:16 untreated 

135 

Dilution 

Spodoptera littoralis Helicoverpa armigera Agrotis ipsilon 

Scotia segetum Mamestra brassicae 

1:1 1:2 1:4 1:8 1:16 

Dilution rate 

XP01broth XP01 F.D. Technical powder 


29 

BIOASSAYS FOR SCREENINGS AND QUALITY CONTROL OF PRODUCTS WITH 

INSECTICIDAL, FUNGICIDAL AND NEMATOCIDAL ACTIVITY, BASED ON PLANT EXTRACTS, 

MICROORGANISMS AND CHEMICAL MOLECULES 

M. Ricci, G. Flek, M. Colli, R. Barcarotti, L. Quattrocchi, S. Coranelli, L. Concezzi, A.P. Fifi, R. De Nicola, M. 

Scribano, M. De Berardinis, F. Citarrei and A. Ragni 

Introduction 


Tel: ++39 – 075 - 895091 Fax: ++39 – 075 – 888 776 E-mail: mricci.bt@parco3a.org 

A bioassay is an experiment where a living organism (insect, plant, phytopathogenic nematode and others) is 

used as test (Robertson J.L. & Preisler H.K.). It is a fundamental tool in the R & D of agricultural products. 

Bioassays are used to discover new molecules and/or toxins, to compare different pesticides, to evaluate the 

feasibility of excipients for formulations, attractants and/or repellents, and for the quality control of products 

ready for the market. Does not exist a universal bioassay: for each purposes a specific bioassy should be used or, 

if not available, should be developed (Ricci et al., 1996). A good bioassay should be cheap, fast, should allow the 

repeatability of the results in different dates and different sites, should discriminate two products with little 

differences, and should predict, as far as possible, the further performance of the product in field conditions. 

Here, are presented a series of bioassays that are used, adapted and developed by the company BioTecnologie 

B.T. S.r.l., for its R&D programs or for services for third parties. 

Bioassays 

Commercial companies that produce and sell bio-products for agricultural use, based on Entomopathogenic 

Nematodes, Fungi, Bacteria and Viruses, have to set up a bioassay unit in order to perform screening programs 

and to carry out quality control of the production and quality control of final products. 

The bioassay should be a compromise between the need to assure scientific results and the need to save time and 

money. 

To support a rational bioassay unit, other sections are needed: Insectary, Plant Pathogen Maintenance and 

Greenhouses. 

Screenings 

Screenings are performed in order to discover new active ingredients or to support the development of products. 

According to the different needs, different bioassays are designed. 

At BioTecnologie B.T., since 1992, several procedures for bioassays have been developed. 

Toxicity bioassays and feeding behavior tests are performed daily for inside and outside (external service) 

research purposes. 

Quality Control of Bio-products for agricultural pest control 

The first duty of the quality control system is to assess what is declared on the label of the product in terms of 

product weight and titer of active ingredient. 

Even if bio-products are based on non-pathogenic (to human beings) microorganisms, the producer has to assure 

that his product is free of pathogenic contaminants (Ricci and Fridlender, 1998). 

Stability is an important parameter in terms of maintenance of the product characteristics during storage and 

transportation. 

The activity is the parameter that shows the ability of the product to control the target insects in real conditions. 

This is the harder parameter to assess because a simple, cheap, fast and reliable laboratory bioassay has to be 

found in order to predict the activity of the product under field conditions 

Insectary 

A multiple-species insect rearing facility for research support on bio-insecticide development is operating at 

BioTecnologie B.T. S.r.l., in Umbria – Italy, since 1992. 


136

Several colonies of Lepidopteran, Coleopteran and Dipteran species have been established and are maintained on 

a regular basis whereas other species are reared on a temporary basis when needed. Standardized rearing 

procedures have been developed that ensure consistent quality of the test insects. 

The following species are currently reared: 

Lepidopteran: 

Agrotis ipsilon��Mamestra 

brassicae, Spodoptera littoralis, S. exigua, Helicoverpa armigera, Lobesia botrana, 

Cydia molestaColeopteran: 

Otiorhynchus sp., Tenebrio molitorDipteran: 

Culex pipiens pipiens 

Plant Pathogen Colony Maintainance 

Several pathogens are maintained regularly in vitro to support the research and development of bio-phytosanitary 

products. 

The following fungi species are currently maintained: 

Fusarium culmorum, F. crookwellense, F. oxysprum dianthii, F. sambucinum, F. solani, F. graminearum, F. 

akuminatum, F. oxysporum, Rhizoctonia solani, Alternaria solani, Botrytis cynerea, Penicillum italicum, 

Cercospora beticola, Phytophthora cinnamoni, Pyranochaeta lycopersici, Phoma spp., Microdochium nivale 

The following phytopathogenic nematode species is currently reared: 

Meloidogyne incognita 

Greenhouses 

Modern and well-equipped greenhouses support the growth of different plant species for insect and 

phytopathogenic nematode rearing, pathogen maintenance and for glasshouse assays. 

The following plants have been grown: 

Corn, Melon, Tomato, Pepper, Cyclamen, Impatient, Tobacco, Celery, Rapeseed . 

Conclusions: 

Due to its facility and its experience, BioTecnologie B.T., is able to design, develop and perform several 

bioassays for the development and the quality control of pesticides and biopesticides. 

References 

Ricci M., Glazer I. and Gaugler R., 1996. Comparison of Bioassays to Measure Virulence of Different 

Entomopathogenic Nematodes. Biocontrol Science and Technology (1996) 6, 235-245 

Ricci M. and Fridlender B., 1998. Quality Control of a Biological Insecticide Based on Entomopathogenic 

Nematodes: NEMA-BIT. At Gent, B: Cost 819 Workshop on Quality Control of Entomopathogenic Nematodes. 

p. 16 and 34-35 

Robertson J.L. &. Priesler H.K, 1992. Pesticide bioassays with arthropods. CRC Press, Boca Raton. 

Pictures 

Bioassay Tray: most of the screenings for micro-organisms 

and molecules against target insects are carried out in this 

particular tray where individual insect is tested on an artificial 

diet 


137

Tenebrio-Assay: researchers are carrying 

on a bioassay 

Phytopathogenic fungi: a bioassay to 

screen micro-organisms and molecules 

active against phytopathogenic fungi 

Insectary: the Lepidopteran room, where different 

lepidopteran species are reared in standard conditions 

138 

Greenhouse: a trial is the greenhouse 



139

30 

ISOLATION OF NEW BIOLOGICAL CONTROL AGENTS FROM UMBRIA REGION 

M. Scribano and A. Ragni 


Tel: ++39 – 075 - 895091 Fax: ++39 – 075 – 888 776 E-mail: aragni.bt@parco3a.org 

Summary 

Samples of soil were collected in two areas of the Umbria region, Valnerina and Oasi di Alviano; the samples 

were used for the isolation of entomopathogenic agents through a selective technique based on the usage of 

bait. There were isolated 13 different samples of entomopathogenic nematodes, 25 entomopathogenic fungi, 27 

sporogenous bacteria and 44 non-sporogenous bacteria. The activities of these microbiological agents were 

verified through in vitro and in vivo tests. 


1. Sampling: 80 soil samples (1 Kg each) were collected till the depth of 20/25 cm from two natural areas of 

Umbria region: Valnerina and Oasi di Alviano. 

2. Baiting with Galleria mellonella 1 : the soil samples were put in containers together with a bait, larvae of the 

Galleria mellonella insect; they were incubated in the dark at 25°C. Periodically the boxes were controlled; the 

dead insects were collected and used later for the isolation of microbiological agents. 

3. Isolation of nematodes: the collection of nematodes was carried out through White-trap 1 : the gummyconsistence 

Galleria cadavers were placed on a filter paper imbibed with Ringer solution in order to allow the 

nematodes to emerge (fig1). The entomopathogenic power of collected nematodes was tested through infections 

of Galleria mellonella larvae 2 : 7 last-instar larvae of Galleria mellonella were treated with 100 nematodes per 

larva. 

4. Isolation of fungi 3 : the hyphae, developed on the surface of the turgid cadavers, were streaked on PDA (potato 

dextrose agar) Petri dishes containing 150 µg/ml of streptomycin. Than the dishes were incubated at 25°C. 

5. Isolation of bacteria: 

Non sporogenous bacteria: the flabby cadavers of Galleria were homogenized in a physiological solution; the 

suspension was plated on NA (nutrient agar) Petri dishes containing 150 µg/ml of nystatin. Than the dishes were 

incubated at 25°C. 

Sporogenous bacteria: an aliquot of the insect cadaver suspension was treated for 12 minutes at 78°C and then 

was streaked on NA. 

The entomopathogenic power of the isolated bacteria towards Mamestra brassicae (Lepidoptera) was verified 

though in vivo tests using an overlay technique bioassay 4 : 32 neonates of M. brassicae were treated with the 

fermented broths of the isolated bacteria. Identification of bacteria was performed with gallery API 50 CHB/E 

Medium. 

Results: 

Only 3 out of 27 of the sporogenous bacteria caused 100% mortality towards Mamestra brassicae. API 50 

CHB/E test showed that two of three active bacteria strains were Bacillus thuringiensis species as confirmed by 

microscopical observation of the sporulating cells (fig.2). B. thuringiensis is the most important microbiological 

agent for control of pest insects 5 . The third strain is being identified.The entomopathogenic power of the 13 

isolated nematode strains were tested through infections of Galleria mellonella; the obtained mortality was high 

although variable, as illustrated in Table1. 

Conclusion 

The study has shown the possibility of isolating samples of micro-organisms useful for the biological control 

from natural habitats. The study will need more screenings in order to evaluate the entomopathogenic power, 

the conditions of use and the possibility of production of the discovered micro-organisms for using them in the 

integrated pest management. 



140

M. Scribano research work was supported by grant of 3A Parco Tecnologico Agroalimentare dell’Umbria, in the 

frame of Umbria Region Training programm (European Social Forum found). 

References 

1 

.K.V. Deseo Kovacs, L. Rovesti. 1992. Lotta biologica contro i fitofagi; Edagricole 

2. 

Bedding, R.A. & Akhurst, R. J. 1975. A simple technique for the detection of insect parasitic rhabditid 

nematodes in soil; 

Poinar, G. O. 1979. In: Nematodes for biological control of insects. CRC Press, Boca Raton, Florida; pp.227. 

3 

Burge, M. N. 1988. Fungi in biological contol systems. Manchester Univ. Press, Manchester & New York; 

pp.296; 

4 

Deseo, K. V. 1991. Batteri entomopatogeni come mezzi di lotta microbiolo gica. Inf. Fitopat. 10, 7-13. 

5 

Li, R. S., Jarrett, P. & Burges, H. D. 1987.– Importance of spores, crystal and delta endotoxin in the 

pathogenicity of different varieties of Bacillus thuringiensis in Galleria Mellonella. J. Invert. Pathol. 50; 277- 

284. 

Figure 1. White-trap with entomopathogenic nematode parasitized insects 


141

Figure 2. Vegative (a) and sporulating cells (b) of B. thuringiensis isolate 

b 

a 

Table1. Mortality of last-instar larvae of G. mellonella 

treated with nematodes isolates at the dose of 

100 nematodes per larva. n=7 


142

Nematode isolate % Mortality 

3 100 

4 40 

9 60 

12 100 

13 100 

14 100 

27 40 

29 55 

38 70 

39 100 

53 100 

58 100 

73 70 


143

31 

PROTEIN CONTENT AND MYCOTOXINS CONTAMINATION IN ORGANIC AND 

CONVENTIONAL WHEAT 

Pietri A., Bertuzzi T., Barbieri G. and Rossi F. 

Istituto di Scienze degli Alimenti e della Nutrizione, Università Cattolica del Sacro Cuore, Facoltà di Agraria, 

Piacenza. 

Introduction 

The importance of organic farming in terms of foods production and consumer confidence is increasing. 

Numerous reports show as a growing number of people believe that organic foods are healthier than 

conventional ones, particularly for the absence of pesticide residue in organic foods. However cereals from 

organic production were sometimes found to have higher mycotoxins content than conventionally produced 

foods, probably because if synthetic pesticide are not used there is a lower crop protection towards parasite 

fungi. However due to some inconsistent results, a clear trend could not be identified. In respect of the proteins 

content, a trend to lower values in organically produced cereals was observed. 

Aim of this work was to verify the content of ocratoxin A (OTA), trichothecenes, ergosterol and crude protein in 

organic and conventional wheat produced in the area of Piacenza (North Italy). 


Thirty-five samples of wheat (20 organic and 15 conventional) were collected during storage and analyzed for 

theirs mycotoxins contamination and crude protein content. OTA was extracted with a mixture of CH3OH:3% 

NaHCO3 water solution(50:50) and purified using an immunoaffinity column (Ochraprep). OTA was determined 

by reversed-phase HPLC with fluorescence detection (λecc=333 nm e λem=470 nm); a Select-B RP-8 column, 

125x4mm (Merck), was employed at room temperature with a mobile phase of acetonitrile:acidified water (2% 

CH3COOH)(41:59) at 1.2 ml/min. The detection limit was 0.010 µg/kg. 

Deoxynivalenol (DON), 3 and 15-acetyl-deoxynivalenol (3-AcDON and 15-AcDON), nivalenol (NIV), HT-2 

and T-2 were determined. These trichothecenes were extracted with acetonitrile:water (84:16) for 90 minutes and 

an aliquot (6 ml) of the filtered extract was purified by Mycosep 227 column (Romer Labs). The final extract 

was derivatised with N-trimethylsilylimidazole and analysed by GC-MS using an ion trap detector. 

Trichothecenes were separated with a RTX-5 capillary column, 30mx0.25mm i.d. (Restek), and quantified by the 

selected ion monitoring technique; the detection limit was 2 µg/kg. 

Ergosterol analysis was performed according to AFNOR (Norme Francaise, 1991). After extraction with KOH 

alcoholic solution, an aliquot (20 ml) of the filtered extract was purified by Extrelut column (Merck). Ergosterol 

was determined by HPLC with UV detection (λ=280 nm); a Superspher Si 60 125x4 mm column (Merck) was 

employed with a mobile phase of n-hexane:isoamyl alcohol=98:2 at 1.0 ml/min. Detection limit was 0.4 mg/kg. 

The N content was determined using the Kjeldahl technique. Statistical analysis of data was performed with the 

statistical package SAS, using the PROC UNIVARIATE to verify the normality of data distribution and PROC 

GLM and PROC NPAR1WAY for analysis of variance. 

Results 

The analyzed samples had a low contamination of mycotoxins, OTA levels were always lower than legal 

threshold of 3 µg/kg and no significant differences were observed between the two types of wheat. The 45% of 

organic samples were contaminated by OTA against a 40% in conventional wheat, however the most 

contaminated sample (0.53 µg/kg) was found in conventional cereals. 

The only detected trichothecene was DON, confirming the hypothesis that it is the most widespread in cereals. 

Only 25% of organic wheat showed a DON contamination (max value = 17.1 µg/kg) while all conventional 

samples were contaminated (max value =219.1 µg/kg). The DON content of organically produced wheat was 

significant lower than conventionally grown one (2.85 µg/kg vs 99.67 µg/kg; P

In our trial organic wheat showed a lower mycotoxins contamination than conventional wheat, on the other hand 

the adoption of organic production system leads to a reduction in crude protein content. Further studies are 

required in order to clarify the effect of organic cultivation system on nutritive value and content of 

antinutritional factor in wheat. 


145

32 

DETERMINAZIONE DI RESIDUI METALLICI NELLE PERE 

E INFLUENZA SULL’IMPATTO AMBIENTALE 

P. Tedeschi 1 , A. Maietti 1 , D. Mazzotta 1 , G. Vecchiati 1 , P. Romano 2 , V. Brandolini 1 

1 Dipartimento di Scienze Farmaceutiche – Università di Ferrara 

2 Dipartimento di Biologia, Difesa a Biotecnologie Agroforestali -Università della Basilicata 

INTRODUZIONE 

La presenza di residui di antiparassitari nelle derrate alimentari sia di origine animale (carni, latte, uova, 

burro, eccetera) sia di origine vegetale (ortofrutticoli freschi, conservati o lavorati industrialmente, vino, olio), 

determina, collateralmente alle preoccupazioni di ordine igienico-sanitario, anche ripercussioni negli scambi 

commerciali, specialmente a livello internazionale. Al fine di limitare il più possibile la presenza dei residui di 

fitofarmaci nelle derrate agricole e ridurre conseguentemente i rischi di intossicazioni acute e croniche per i 

consumatori, si stanno studiando e valutando sistemi di coltivazione che consentano l’applicazione delle più 

moderne tecniche di "produzione integrata" e di “produzione biologica”. 

Per quanto riguarda la difesa dalle crittogame i formulati a base di rame sono prodotti chimici ammessi dai 

disciplinari di produzione integrata e biologica. E’ stato dimostrato che il rame svolge un ruolo chiave in 

numerose funzioni biochimiche e metaboliche degli organismi ma un suo accumulo può provocare fenomeni 

tossici gravi. La dieta occidentale contiene abitualmente una quantità di rame più che sufficiente a ricoprire il 

fabbisogno giornaliero che per un adulto sano, di età compresa tra i 23 e i 50 anni, è di circa 2 mg (1,5-3 mg). 

Ne sono particolarmente ricchi i legumi, il pesce, i crostacei, la carne, i cereali e le noci.. Da studi epidemiologici 

condotti precedentemente, risulta che per pazienti con elevati livelli di rame (> 1.43 mg/l) il rischio relativo di 

morte per malattie cardiovascolari e tumori è quattro volte più elevato che nei soggetti con livelli normali 

(

per "strippare" i vapori nitrosi residui mediante saturazione dell'ambiente con O2. Si scalda nuovamente ed 

infine si lascia definitivamente raffreddare: si ottengono così soluzioni limpide, più o meno incolori. 

Le analisi sono state effettuate in triplo e per la lettura dei campioni è stato utilizzato uno spettrofotometro per 

assorbimento atomico Perkin Elmer 1100B dotato di una lampada a catodo cavo, a singolo o multielemento, di 

tipo Intensitron TM Perkin Elmer e di un bruciatore a premiscelazione al Titanio, a fenditura singola di 10 cm, 

per fiamma ad aria /acetilene (2145/2400°C SIO S.p.A. Milano). 

Come prova di controllo (bianco) è stata eseguita una mineralizzazione con i soli reagenti al fine di osservare 

loro eventuali interferenze nella determinazione quantitativa dei metalli presenti. 


La prima parte del lavoro è stata incentrata sulla messa a punto del metodo di mineralizzazione ed analisi dei 

diversi campioni oggetto della ricerca. Per stimare gli eventuali effetti dovuti ad interferenze da matrice sono 

state eseguite numerose prove di recupero basate sul metodo delle aggiunte. In particolare ogni matrice 

considerata (terreno, foglie e pere) è stata addizionata (fortificata) con soluzioni standard di elemento in esame e 

tali da determinare sui campioni incrementi noti e pari a 0,1 e 1 µg/g. 

Dall’esame complessivo dei risultati ottenuti riportati in tabella 1, si può evidenziare come tutti i valori di 

recupero siano compresi fra il 97,8% e il 102% per cui si può affermare che la metodologia in esame rientra 

pienamente nei criteri di attendibilità previsti dalle raccomandazioni IUPAC. 

Nella tabella 2 sono riportate le concentrazioni medie dei diversi metalli presenti nei campioni di foglie e terreno 

raccolti in tempi diversi all’interno delle parcelle sperimentali trattate secondo i criteri di “Difesa Biologica” e di 

“Difesa Integrata”. 

Tabella 1. Valori medi di recupero (% ± SD) determinati per i diversi metalli presenti nelle matrici analizzate e 

ottenuti con il metodo delle aggiunte a diversi livelli di concentrazione ( µg/g). 

================================================================== 

Recupero (% ±SD) 

--- ----------------------------------------------------------------- ------------------------ 

Terreno Foglie Pere 

Metallo --------------------------------------------------------------------------------------------- 

Concentrazioni addizionate ( µg/g) 

-- ------------------------------------------------------------------------------------------- 

0.1 1.0 0.1 1.0 0.1 1.0 

___________________________________________________________________________ 

Rame 100.5±1.2 100.3±1.7 98.2±2.1 99.6±1.5 98.2±1.9 100.1±1.6 

Ferro 99.8±2.3 97.9±3.1 101.0±1.9 98.0±2.7 99.2±2.8 101.1±2.1 

Zinco 100.3±2.1 98.2±2.8 96.4±3.8 99.1±2.7 101.2±2.8 100.0±1.4 

Magnesio 98.3±1.4 99.2±2.2 100.2±3.1 98.3±2.8 102.0±2.5 99.9±2.9 

Nichel 102.0±2.6 98.4±3.0 100.4±2.8 98.2±3.1 98.6±3.2 98.1±3.4 

Cromo 100.5±3.0 102.1±2.8 99.2±3.1 98.7±2.4 97.8±2.7 100.4±3.3 

================================================================== 

L’ipotesi nulla H0 sull’intercetta è stata valutata per p=0,01. Il limite di rivelabilità è stato calcolato secondo le 

raccomandazioni IUPAC per p=0,01. 

Il principale interesse della ricerca era incentrato sulla rilevazione delle concentrazioni di rame che si sarebbero 

determinate in relazione al numero e al tipo di trattamenti eseguiti, e da un primo gruppo di metalli considerati 

indispensabili: ferro, zinco e magnesio. Da ciò e sulla base dei risultati ottenuti, si può osservare che il contenuto 

tali metalli nelle foglie e terreno aumenta considerevolmente dalla prima alla seconda raccolta nei campioni 

trattati e rispetto al testimone, mentre nella terza raccolta l’aumento è molto contenuto e non si differenzia 

significativamente per le due modalità di difesa. Il testimone, costituito da piante che non hanno subito 

trattamenti chimici, presenta ridotti contenuti di rame. 

Le differenze riscontrate non sono significative nell’ambito di ciascuna raccolta, tuttavia, per il ferro, si nota che 

i campioni del testimone presentano costantemente concentrazioni superiori a quelle dei campioni trattati. Nichel 

e cromo sono rilevabili in concentrazioni limitate in tutte le tesi e permangono pressoché costanti per tutto l’arco 

della sperimentazione. 


147

Tabella 2. Concentrazioni medie (µg/g) dei residui di metalli presenti nelle foglie e nei terreni provenienti dai 

campi sperimentali di difesa biologica ed integrata. F = Foglie; T = Terreno. 

Difesa Prelievo Rame Ferro Zinco Magnesio Nichel Cromo 

F T F T F T F T F T F T 

Biologica 1° P 31,5 90,2 53,6 10983 n.d. 70,0 3505 5815 2,7 41,6 0,6 10,3 

Integrata 1° P 45,1 93,6 58,5 9076 n.d. 104,0 3471 6591 3,1 46,7 0,4 13,5 

Testimone 1° P 18,9 77,0 63,4 11329 n.d. 57,1 3741 6214 4,0 37,9 0,8 10,0 

Biologica 2° P 424,2 131,2 73,4 23010 45,7 127,0 3481 6289 3,9 42,5 1,1 10,3 

Integrata 2° P 401,0 125,7 69,4 22795 48,5 121,4 3192 6646 3,5 43,5 1,1 9,6 

Testimone 2° P 9,7 103,6 87,3 23779 46,8 131,4 3373 6507 5,1 43,3 1,0 11,3 

Biologica 3° P 462,3 97,7 75,7 22367 60,6 118,8 3811 6736 1,4 44,0 1,2 12,0 

Integrata 3° P 460,0 93,3 99,6 24114 55,2 109,4 3305 7078 2,2 45,5 1,4 13,8 

Testimone 3° P 18,6 91,4 100,1 24300 46,8 124,9 3732 7282 1,0 46,0 2,3 12,2 


148

L’ultima parte della ricerca è stata dedicata alle determinazioni sui frutti. I risultati ottenuti sono riportati in 

tabella 3. Per quanto riguarda gli elementi indispensabili ferro, zinco e magnesio si può notare che sono presenti 

a valori pressoché costanti e senza notevoli differenze tra i due sistemi di difesa. Unica eccezione un leggero 

aumento delle concentrazioni del magnesio nel secondo prelievo specie utilizzando il sistema di difesa integrata. 

Per il cromo va sottolineato come esso sia presente in concentrazioni molto ridotte in tutte le tesi e in tutte e tre 

le raccolte mentre per il nichel i valori sono risultati sempre al di sotto del limite di rivelabilità dello strumento. 

Tabella 3. Concentrazioni medie (µg/g) dei residui di metalli presenti nei 

frutti provenienti dai campi sperimentali di difesa biologica ed integrata. 

Difesa Prelievo Pere 

Rame Ferro Zinco Magnesio Nichel Cromo 

Biologica 1°P 2,64 3,04 1,40 70,85

CONCLUSIONI 

La ricerca intrapresa, anche se limitata ad una sola annata agraria per cui sono auspicabili ulteriori conferme, ha potuto 

mettere in evidenza alcuni punti di sicuro interesse. Innanzitutto vanno evidenziati i risultati relativi alle determinazioni 

eseguite sui frutti che, rappresentando la parte edule e commerciale, interessano principalmente i produttori e i 

consumatori. 

I risultati ottenuti evidenziano un quadro positivo; in particolare, si è dimostrata valida la difesa biologica per quanto 

riguarda gli aspetti igienico-sanitari e di conservazione. Confrontando infatti le percentuali di frutti colpiti alla raccolta 

si può osservare che il valore più basso è quello relativo alla difesa biologica (46,75%) contro il 54,87% della difesa 

integrata e l’81% del testimone. Per ciò che concerne invece i residui di rame presenti nel terreno, nelle foglie e frutti 

trattati secondo le modalità di difesa biologica e integrata, le relative concentrazioni risultano sempre inferiori al limite 

di tolleranza previsto dalla vigente legislazione e che per la frutta è fissato in 20 µg/g. 

Da queste indicazioni si può quindi dedurre che le preparazioni a base di rame possono costituire un valido metodo di 

difesa anticrittogamica essendo selettive ed efficaci anche a basse concentrazioni oltre ad essere di sicuro interesse data 

la loro limitata pericolosità per ciò che concerne gli effetti antropotossici. 

La prosecuzione della ricerca potrà fornire ulteriori elucidazioni su altre colture ma soprattutto mettere in evidenza in 

un programma colturale completo quali siano i rischi/benefici dovuti all’applicazione delle più attuali strategie di difesa 

al fine di migliorare le produzioni sia dal punto di vista quantitativo che qualitativo andando quindi maggiormente 

incontro alle pressanti richieste dei consumatori. 

Bibliografia 

- Aceto M., Abollino O., Bruzzoniti MC., Mantasti E., Sarzanini C., Malandrino M. (2002). Determination of metals in 

wine with atomic spectroscopy (flame-Aas, GF-AAS and ICP-AES); a review. Food Addit Contam. 19: 126-33. 

- Anderson R.A., Bryden N.A., Polansky M.N., Deuster P.A. (1995). Acute exercise effects on urinary losses and 

serum concentration of copper and zinc of moderately trained and untrained men consuming a controlled diet. Analyst 

120: 867-870. 

- Brandolini V., Tedeschi P., Capece A., Maietti A., Mazzotta D., Salzano G., Paparella A., Romano P. (2002). 

Saccharomyces cerevisiae wine strains differing in copper resistance exhibit different capability to reduce copper 

content in wine. World Journal of Microbiology & Biotechnology. 18: 499-503. 

- Chow P.Y.T., Chua T.H., Tang K.F., Ow B.Y. (1995). Dilute acid digestion procedure for the determination of lead, 

copper and mercury in traditional chinese medicine by atomic absorption spectrometry. Analyst 120, 1221-1223. 

- Epsein L., Bassein S. (2001). Pesticide applications of copper on perennial crops in California, 1993 to 1998. J. 

Environ Qual. 30(5) : 1844-1847. 

- Hamey PY., Harris CA. (1999). The variation of pesticide residues in fruits and vegetables and the associated 

assessment of risk. Regul. Toxicol. Pharmacol. 30: 34-41. 

- Tahan J.E., Sànchez J.M., Granadillo V.A., Cubillàn H.S., Romero R.A. (1995). Concentration of total Al, Fe, Hg, 

Na, Pb and Zn in commercial canned seafood determined by spectrometric means after mineralization by microwave 

heating. J. Agric. Food. Chem. 43: 910-915. 


150

33 

INVESTIGATIONS ON THE BACTERICIDAL ACTIVITY OF SOME NATURAL PRODUCTS 

Varvaro L., M. Antonelli, G. M. Balestra, A. Fabi, D. Scermino e G. Vuono 

Dipartimento di Protezione delle Piante, Università degli Studi della Tuscia, 

Via S. Camillo de Lellis, 01100, Viterbo, Italy 

The Council Regulation (EEC) no. 2092/91 shows the rules on organic production in agriculture to ensure both 

consumers and producers. The products for plant protection (copper compounds, propolis and sodium silicate) are listed 

in the Annex II part B of this regulation. The main chemicals used in organic agriculture to control bacterial plant 

diseases are cupric compounds. The last EEC Regulation (Reg. no. 473/2002) limits the use of copper and force the 

researchers to study new way in controlling bacterial diseases. The utilization of natural substances, such as propolis 

and sodium silicate, is one way susceptible to be investigated in order to better know their bactericidal or bacteriostatic 

effect on some host pathogen interactions. 

In particular, the activity of propolis and sodium silicate, alone or in association with different copper 

compounds, were in vitro studied toward a large number of phytopathogenic bacterial strains, both Gram positive and 

Gram negative. Furthermore, some in vivo tests were performed against some bacterial diseases of tomato, by spraying 

commercial products containing propolis and sodium silicate and on hazelnut using copper compounds. In vitro results 

show that propolis is active against the growth of the most of Gram positive bacterial strains, even at relatively low 

concentrations, while it is very poorly active on almost all essayed Gram negative ones. Antagonistic action of sodium 

silicate resulted evident only at high concentrations; in particular a certain in vitro inhibition was detected on 

Pseudomonas syringae pv. tomato the agent of bacterial speck of tomato, but it was not confirmed by in vivo tests. An 

inhibitory effect on epiphytic populations of Xanthomonas vesicatoria, the agent of bacterial spot of tomato, was 

observed till to 7 days after the treatment. Sodium silicate was able to control also epiphytic populations of Clavibacter 

michiganensis subsp. michiganensis, the agent of bacterial canker of tomato. The in vitro and in vivo studies on the 

effect of different copper compounds in controlling phytopathogenic bacteria showed some interesting results. Copper 

oxychloride and new chemicals, like tri-basic copper sulphate or pentahydrate copper sulphate, were sprayed on 

hazelnut plants showing dieback symptoms. The obtained results confirmed the preventive bactericidal effect of copper 

oxychloride against the pathogens and, moreover, pointed out the good effectiveness of the pentahydrate copper 

sulphate. In fact, even if this last compound is characterized by a copper concentration five time lower than the copper 

oxychloride, and although it has been sprayed at reduced field-doses, it gave the same results in controlling the hazelnut 

disease. 

All results showed that propolis and sodium silicate could be useful in controlling epiphytic bacterial 

populations of some phytopathogenic bacteria on tomato, especially in organic agriculture. On the other hand, copper is 

confirmed to be used to diminish the populations of phytopathogenic bacteria and to reduce in such a way the disease 

diffusion. The opportunity to successfully use chemical products characterized by low copper concentration is important 

to safeguard the environment and the human health. 


151

34 

RICERCA SU INSETTICIDI UTILIZZABILI SECONDO IL METODO DI PRODUZIONE BIOLOGICO DI 

PRODOTTI AGRICOLI 

Fabio Molinari – Piero Cravedi 

Istituto di Entomologia e Patologia vegetale – Facoltà di Agraria U.C.S.C. – Piacenza – e-mail entomo@pc.unicatt.it 

Introduzione 

La protezione del pesco con il metodo biologico di produzione è attuata da alcuni anni con vari prodotti ad azione 

antiparassitaria di cui non è ben nota l’efficacia. 

Varia attività sperimentale è stata condotta negli anni con finalità non sempre aderenti alle esigenze del metodo. 

La valutazione dell’efficacia dei prodotti antiparassitari non può essere fatta prescindendo dal metodo complessivo di 

coltivazione che è determinante nell’influenzare la sensibilità delle piante agli attacchi parassitari. La carenza di 

informazioni sull’argomento non consente di avere un quadro preciso del ruolo che certi prodotti possono svolgere nella 

difesa delle colture (pesco e di melone nella presente ricerca). L’obiettivo che ci si propone di ottenere dati che, tenendo 

conto della filosofia del metodo della produzione biologico, risultino valutabili secondo criteri scientifici. 

l’Istituto di Entomologia di Piacenza svolge dal 1998 una Ricerca su insetticidi utilizzabili secondo il metodo di 

produzione biologico di prodotti agricoli nell’ambito del Progetto: Difesa delle produzioni in agricoltura biologica 

promosso dal MINISTERO PER LE POLITICHE AGRICOLE. 

La ricerca si inserisce sulla “produzione integrata” nei pescheti che da anni l’Istituto di Entomologia di Piacenza sta 

conducendo in varie regioni italiane in collegamento con le principali istituzioni di ricerca europee. 

Materiali e Metodi 

Sono state effettuate prove parcellari per determinare l’efficacia nel controllo di insetti di alcuni prodotti per 

l’agricoltura biologica. 

Inizialmente i prodotti da sperimentare erano quassine ed oli vegetali su afide verde del pesco e su Aphis gossypii su 

cucurbitacee, ryania su Cydia molesta su pesco. 

In corso di svolgimento, anche in relazione all’interesse mostrato da parte dei produttori interpellati per l’esecuzione 

della sperimentazione, sono state inserite prove con Azadiractina in sostituzione di quelle con oli vegetali, che non è 

stato possibile allestire per mancanza di interesse da parte di tecnici ed agricoltori; in fase avanzata sono state inserite 

prove per la valutazione di ryanodine contro Cydia funebrana. 

Pertanto le prove effettuate nel progetto sono state: 

Quassio contro M. persicae su pesco (su reinfestazioni dopo la fioritura) 

Quassio contro A. gossypii su melone (anguria) 

Ryania contro C. molesta su pesco 

Ryania contro Cydia funebrana su susino 

Azadiractina 3 contro C. molesta su pesco 

Azadiractina 3 contro M. persicae su pesco 

Azadiractina 3 contro A. gossypii su melone (anguria) 1 - Polvere micronizzata (400 g/hl) 

2 - polvere setacciata 180 mm, con contenuto di ryanodine 0,1%. (600 g/hl) 

3 - Oikos, Sipcam (100 g/hl) 

Il progetto prevede una prima fase dedicata alla determinazione dell’efficacia, mentre nelle fasi successive verranno 

ricercati i criteri per un’utilizzazione ottimale dei prodotti. 

Lo schema di base è stato il seguente: 

Aphis gossypii. Prove parcellari secondo lo schema proposto dall’EPPO su cotone. Trattamento effettuato all’inizio 

dell’arrivo degli insetti alati e ripetuto dopo 7 giorni. 

Myzus persicae. Prove parcellari. 1998-1999: trattamenti effettuati sulle reinfestazioni in post-fioritura quando l’azione 

degli antagonisti inizia a manifestarsi e potrebbe essere influenzata negativamente da trattamenti a base di piretro o 

rotenone. 

2000-2001: concentrando maggiormente l’attenzione sull’azadiractina, sono stati valutati gli effetti di trattamenti pre e 

postfiorali. 

Cydia molesta. Prove parcellari. Trattamenti effettuati sulle larve della seconda generazione per meglio evidenziare il 

livello di efficacia. 

Cydia funebrana. Prova a parcelloni sulle larve di seconda generazione. I trattamenti sono stati ripetuti 3 volte (2 per 

ryania) a distanza di 8-10 giorni. 

Risultati 


152

Vengono qui riportati i risultati di alcune prove rappresentative. Aphis gossypii: le scarse infestazioni nei campi 

sperimentali non hanno consentito di trarre indicazioni chiare. Nella prova di Castellazzo Bormida (a) 

Myzus persicae: le prime prove hanno evidenziato una limitata attività del quassio sulle reinfestazioni, mentre più 

interessante è risultata l’azione di azadiractina; buona l’efficacia prefiorale, chiaramente migliorata dalla ripetizione del 

trattamento in postfioritura (b). 

Cydia molesta. Sia azadiractina sia rianodine hanno mostrato una attività variabile, ma complessivamente tale da 

suggerire ulteriori approfondimenti, sia sulle modalità applicative, sia sulle formulazioni; in particolare le rianodine 

hanno presentato alcune difficoltà in fase di preparazione e di distribuzione, e nonostante ciò hanno fornito risultati 

quasi sempre in linea, e talvolta superiori a quelli dei preparati a base di Bacillus thuringiensis.. Un esempio è riportato 

in figura c. 

Cydia funebrana. E’ stata effettuata una sola prova nel 2002 su parcelloni con 2 repliche. Dal grafico (d) si può 

osservare che nelle parcelle trattate con ryania e azadiractina si è registrato un calo nell’infestazione, mentre in quelle 

trattate con Bacillus thuringiensis una scarsa azione può essere evidenziata solo dalla riduzione dell’incremento del 

fitofago, con un risultato finale comunque non differente rispetto al testimone. 


153

35 

FUNGICIDES AND HEAVY METALS IN WINE SAMPLES PRODUCED FROM EXPERIMENTAL 

GRAPES SUBJECTED TO DIFFERENT PESTICIDES TREATMENTS 

Salvo Francesco, Saitta Marcello, Di Bella Giuseppa, La Pera Lara, Dugo Giacomo 

Dipartimento di Chmica Organica e Biologica Università di Messina Salita Sperone, 3190166 S. Agata -Messina 

Wine is a wildely consumed beverage with thousand years traditions, so an accurate quantitative knowledge of 

the presence of toxic elements as pesticides and heavy metals, is very important from both toxicological and enological 

point of view. The use of pesticides is closely associated with viticulture (1). Many factors influence the presence of 

heavy metals in wine: type of soil, wine-processing equipment, vinification methods (2). The purposes of this work are 

both to quantify pesticides residues and trace metals in samples of Italian wines, and to study the possible correlation 

between the presence these contaminants (3). 

Wine farms that comply with the Rule Cee 2078/92 for wine grape defense, have to respect all the provided 

restrictions regarding to the use of pesticides. 

To control the growth of powdery mildew (Uncinula necator), the use of sulfur, azoxystrobin, dinocap, 

fenarimol, penconazole, quinoxyfen, is allowed. Mildew epidemic weakens the vines, causing huge production losses 

and negatively influencing the quality of wines. Also an incorrect use of fungicide and frequency of their applications, 

maight alter the quality of the final product. 

The determination of fungicide residues in wine is well documented, even if the reported extraction procedures 

are complex and expensive; meanwhile there is a lack of available data regarding to the extraction of dinocap residue. 

In this work a quick chromatographic method for the simultaneous extraction of azoxystrobin, dinocap, fenarimol, 

penconazole, quinoxyfen residues in wine samples is described (4). 

The same samples of wine were subjected to trace metal determination as Cd (II), Cu (II), Pb (II) and Zn (II). 

Heavy metals dosage was carried out by derivative potentiometric stripping analysis (3), which allowed to execute the 

determinations in a short time directly on the wine sample appropriately acidified. The presence of Cd (II), Cu (II), Pb 

(II) and Zn (II) in white and red wine sample, was correlated to the treatments applied on grapes. 

Samples description 

All the samples of white and red wine were produced in the crop year 2000-2001.White wines came from Sicily 

and Campania red wine from Tuscany. Sicilian wines were produced from Inzolia and Carricante grape variety; wines 

from Campania were produced Fiano d’Avellino grape variety. Red wines from Tuscany were vinified from Sangiovese 

grape variety, Morellino clone. 

The fungicide treatments were carried out on the end of blossoming and maturation; they were used at the doses 

recommended by the manufacturers and were sprayed with a manual sprayer every 7-12 days (9 treatments) The last 

treatments was executed about 28 days before the grape harvest. Only dinocap, which is generally used to start the 

defence strategy, was applied 3 times. A random-block scheme was used with four replications for each test, and each 

block contained 60 plants. 

HRGC analysis 

A HRGC 5300 mega Series Carlo Erba Instruments gas chromatograph was used for determination of five 

fungicides. It was fitted with an electron-capture detection (ECD) system and split-splitless injector, connected to a 

Hewlett Packard 3394 A reporting integrator. 

A SPB-5 fused – silica column (30 m x 0.25 mm i.d.) was employed, with 5% diphenyl 95% dimethyl siloxane 

as liquid phase (film thickness 0.25 µm) (Supelco). The injector and detector were operated at 250° C and 280°C, 

respectively. 

The sample (1 µL) was injected in the split mode (1:3), and the oven temperature was programmed as follows: 

200°C (1 min) raised to 270°C (5°C/min), held for 20 min. Helium was the carrier gas and nitrogen the make-up gas 

both at 150 Kpa. 

Ten grams of wine was collected in an Erlenmeyer flask with a teflon-lined screw-cap. A 20 mL volume of 

hexane-ethyl acetate (9:1, v/v) was added, and the mixture was put in a sonicator (Transsonic T420, Elma) for 30 min. 

A 2 mL volume of supernatant was evaporated under nitrogen flow; 0.2 mL of the internal standard solution 

(bromophos-methyl 1.00 mg/L) was added, and the mixture was directly analyzed by gas chromatography. No clean-up 

was necessary because there were no interference in the chromatogram. 

Previously, a blank assay was employed to check for the absence of residuals in wine. Untreated samples of wine 

were fortified with 0.01, 0.1 and 1.0 mg/Kg of fungicides. The samples were allowed to equilibrate for 60 min prior to 

extraction, and were processed according to the above procedure. The recovery assays were replicated 3 times: 

recoveries ranged fro 75.9 to 121.5 %. The detection limits were evaluated as the peak height that is two times respect 

to the bottom noise. For azoxystrobin, fenarimol, penconazole and quinoxyfen the calculated detection limit is 5 pg/µL; 


154

while for dinocap is 10 pg/µL. For the determination of sulfur residue, a HRGC Dani 86.10, fitted with a FPD Dani 

86/70 detector and a Mega 68 fused Silica column, were used; the detection limit was 2 ng/µL. 

Derivative potentiometric stropping analysis 

For metals determination an ION 3 Steroglass potentiometric stripping analyzer equipped with a conventional 

three electrodes cell, was used. The working electrode was a glassy carbon one covered with a thin mercury film. 

Before each analysis an appropriate volume of wine was treated with HCl 2M lowering the pH to 2, in order to ensure 

that all metals present remain in unbound –free forms. Cd (II), Cu (II) and Pb (II) were determined simultaniously, their 

oxidation potential were respectively –590 mV, -185 mV and –400mV. Zn was separately determined, its oxidation 

potential was –950 mV. Quantitative analysis was always carried out by the method of standard additions. 

RESULTS and CONCLUSION 

The residues found in all wines were below the limits that Italian law (D.M. 19 may 2000) provides for grapes 

and wines. Particularly no quinoxyfen residues were found in the wines; also in wines originated from treated grapes, 

no dinocap residues were found probabily because of an inferior number of tratments applied on vines (table 1). 

Maximum accetable levels of Cd (II), Cu (II), Pb (II), and Zn (II) in wines, has been estabilished by the Italian 

Republic (5) and by the European Commission (6) respectively at 100 

ng L -1 , 1000 ng L -1 , 300 ng L -1 , and 500 ng L -1 . As tables 2 a-c evidence that the metals content of the studied wines was 

always under the acceptable levels, particularly Cd was not found in any sample. The content of Cu, determined in 

wines coming from the three regions ranged from 10.5 to 415.7 ng mL -1 and was not influenced by fungicides 

treatments applied on grapes. The concentration of Pb spanned from 3.6 to 17.4 ng mL -1 and was regularly influenced 

by pesticides treatments (fig. 1): the highest amounts were extimated for samples treated only with sulfur and with 

azoxystrobin while the lowest were found in wines trated with quinoxyfen and dinocap. The level of Zn was in the range 

21.6-263.0 ng mL -1 and showed a regular behaviour respect to the treatments (fig.2) only in white wines: the highest 

amounts were extimated for samples treated with fenarimol and azoxystrobin, and as already observed, the lowest in 

samples treated with dinocap. For red wines the highest Zn contents were evaluated in samples treated with azoxstrobin 

and in the one trated with sulfur, the lowest in the sample treated with fenarimol. 

LITERATURE 

1. Fernadez-Cornejo, J. Environmental and economic consequences of technology adoption: IPM in 

viticulture. Agricultural Economics 1998, 18, 145-155. 

2. Golimowsky, J.; Valenta, P.; Wolfgang-Nurnberg, H. Toxic Trace Metals in Food II. A Comparative 

Study of The Levels of Toxic Trace Metals in Wine by Differential Pulse Anodic Stripping Voltammetry and 

Electrothermal Atomic Absorption Spectrometry. Z.Lebensm. Unters. Forsh. 1979 b, 168, 439-443. 

3. Salvo, F.; La Pera, L.; Di Bella, G.; Nicotina, M.; Dugo, G.mo. Influence of mineral and organic 

pesticides treatments on Cd (II), Cu (II) Pb (II) and Zn (II) content determined by Derivative Potentiometric Stripping 

Analysis in Italian Red and white wines. J. Agric. Food Chem. 2003 in press 

4. Dugo, G.mo; Visco A.; Saitta M.; Vinci, V.; Di Bella G.Dosaggio di quinoxyfen su prodotti vitivinicoli 

siciliani. Vigne vini, 2001, 91, 7-8. 

5. Repubblica Italiana; Caratteristiche e Limiti di alcune Sostanze nel Vino. 1986, december D.M. 29. 

6. European Commission; 1997 Doc III/5125/95/ Rev.3. 

Table 1: Fungicide residues (µg /Kg + s.d.) in wine. 

azoxystrobin dinocap fenarimol penconazole quinoxyfen 

Sicily 21±6 n.d. 13±2 10±1 n.d.* 

Campania 5±1 n.d. 6±2 8±2 n.d.* 

Tuscany 12±2 n.d. 70±6 12±1 n.d.* 

n.d. < 10 µg /Kg 

n.d.* < 5 µg /Kg 


155

Table 2 a, b, c: Cd (ng mL -1 ), Cu (ng mL -1 ), Pb (ng mL -1 ) and Zn (ng mL -1 ) concentrations in 8 samples of wines 

from Sicily (a), 8 from Campania (b), and 8 from Tuscany (c) : one of each group was treated with water only (blank), 

the others were subjacted to different organic and inorganic pesticides treatment. Each value was the mean of three 

determinations. 

(a) 

Samples Treatments Cd Cu 

Pb 

Zn 

Blank Water n.d 20.6±0.7 9.0±0.9 21.6±1.1 

ORGANIC TREATMENTS 

1 Quinoxifen n.d 63.3±1.8 10.3±1.1 23.7±0.8 

2 Fenarimol n.d 27.8±1 11.4±0.9 53.1±0.3 

3 Azoxystrobin n.d 26.8±0.8 16.7±1.4 52.6±1.3 

4 Dinocap-Penconazole n.d 23.2±0.7 9.6±0.8 26.7±0.6 

5 Dinocap n.d 24.4±1.2 13.0±1.3 21.6±1.0 

mean ± sd 33.1±17.0 12.2±2.8 35.5±15.9 

INORGANIC TREATMENTS 

6 S WP n.d 36.1±1.2 13.8±1.0 47.2±1.6 

7 S DP n.d 29.4±1.3 26.0±1.3 53.7±2.0 

mean ± sd 32.8±4.7 19.9±8.6 50.5±4.6 

total mean ± sd 31.5±13.7 13.7±5.6 37.4±15.3 

n.d.= not detectable < 1.5 ng mL -1 

S WP= sulfur wettable powder 

S DP=sulfur dry powder 

(b) 

Samples Treatments Cd 

Blank Water n.d 31.0±0.8 3.6±0.3 77.6±2.0 


1 Quinoxifen n.d 185.3±5.9 4.2±0.2 111.9±1.7 

2 Fenarimol n.d 184.2±2.7 5.1±0.3 205.5±4.3 



5 Dinocap n.d 72.4±1.0 4.1±0.4 142.1±2.6 

mean ± sd 178.8±64.5 6.2±2.9 133.9±79.3 


6 S WP n.d 110.8±4.0 12.6±0.2 178.8±2.5 

7 S DP n.d 174.6±4.0 15.4± 0.8 176.6±2.9 

mean ± sd 142.7±45.1 14.0±2.0 177.7±1.6 

total mean ± sd 151.1±72.7 7.8±4.9 150.3±47.2 

(c) 

Samples Treatments Cd 

Blank Water n.d 10.5±0.5 5.0±0.4 105.0±2.9 


1 Quinoxifen n.d 415.7±12.5 12.6±0.9 263.0±8.0 

2 Fenarimol n.d 103.3±2.3 14.4±1.0 166.7±6.3 



5 Dinocap n.d 325.2±7.3 9.5±0.7 152.2±5.4 

mean ± sd 304.4±128.7 12.6±3.5 172.9±58.0 


6 S WP n.d 20.1±2.3 17.2±0.2 266.9±9.1 

7 S DP n.d 61.4±3.3 16.6±1.0 257.8±8.9 

mean ± sd 40.8±28.2 16.9±0.5 262.4±6.4 

total mean ± sd 198.4±169.3 12.7±4.5 186.8±68.2 


156 

Cu 

Cu 

Pb 

Pb 

Zn 

Zn

[Pb](ng/ml) 

Figure 1: Pb content of wines samples from different treatments 

[Zn] (ng/ml) 

30,0 

25,0 

20,0 

15,0 

10,0 

5,0 

0,0 

300,0 

250,0 

200,0 

150,0 

100,0 

50,0 

0,0 

Water 

Water 

Quinoxiphen 

Quinoxiphen 

Fenarimol 

Fenarimol 

Azoxistrobin 

Dinocap-Penconazole 

Azoxistrobin 

Dinocap-Penconazole 

Figure 2: Zn content of wines samples from different treatments 

157 

Sulfur WP 

Dinocap ( Quinoxyfen) 

Sulfur WP 

Dinocap (Quinoxyfen) 

Sulfur DP 

Sulfur DP 

Sicily 

Campania 

Tuscany 

Sicily 

Campania 

Tuscany 

Sicily 

[Pb](ng/ml) 

Campania 

Tuscany 


36 

NEW α-ELICITIN ISOFORMS FROM Phytophthora hybernalis AS PROTEIN ELICITORS FOR A 

POTENTIAL EMPLOYMENT IN THE BIOLOGICAL PEST MANAGEMENT 

R. Capasso*, A. De Martino*, G. Cristinzio**, A. Di Maro*** and A. Parente*** 

*Dipartimento di Scienze della Pianta, del Suolo e dell’Ambiente, Università degli Studi di Napoli “Federico II”, 

Portici, Italy. E-mail: capasso@unina.it 

** Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi di 

Napoli “Federico II”, Portici, Italy. 

*** Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli, Caserta, Italy. 

The microbial naturally occurring compounds which are able to induce plant defence mechanisms against 

phytopathogenic fungi and bacteria are named elicitors and could be consequently employed in strategies for the 

biological control of plant diseases. 

The genus Phytophthora produces protein elicitors, named α- and β-elicitins, which show a relative molecular weight 

of 10 KDa and 98 amino acids. Their characteristic biological activities are the induction of a hypersensitive response 

(HR) and systemic acquired resistance (SAR) in tobacco with the formation of phytoalexins, PR proteins and salycilic 

acid; moreover, they induce protection against phytopathogenic microorganisms in tobacco and in some species of 

Brassicaceae 1 . 

In the present communication we describe the isolation and the preliminary data on the chemical characterization and 

elicitor activity on tobacco (Nicotiana tabacum L., cv Rustica) of three new elicitin isoforms H-α1, H-α2 e H-α3, 

produced in the culture filtrates of an isolate of Phytophthora hybernalis, the causal agent of rot of citrus fruits, isolated 

from diseased plants in the sorrentina peninsula. 

The three isoforms have been isolated by four purification steps, of which the final step was represented by a 

chromatographic purification on a reverse-phase column (RP-18) eluted at low pressure with a gradient of wateracetonitrile. 

It was determined their absolute molecular weight by MALDI Mass Spectrometry, obtaining values of 10239.24 for Hα1, 

10235.94 for H-α2 and 10244.28 uma for H-α3. 

In studies of elicitor activity carried out testing the hypersensitive response on tobacco, the three isoforms showed 

different intensity responses, that is H-α1 proved to be the most active, followed by H-α2, whereas H-α3 proved to be 

inactive. 

Studies will be carried out on the determination of their primary structure and elicitor effects on other plants of 

agricultural interest. The very low dose required for their positive biological effects on tobacco, their protein nature 

and the no toxicity of the elicitins for adults 2 , render these new compounds and those previously described 1,3,4 very 

interesting for investigations on their possible use in biological pest management programs. 

REFERENCES 

1. Ponchet, M., Panabieres, F., Milat, M.-L., Mikes, V., Montillet, J.-L., Suty, L., Triantaphylides, C., 

Tirilly, Y., Blein, J.-P., 1999. Are elicitins cryptograms in plant-Oomycete communications? Cell Mol. 

Life Sci. 56, 1020-1047. 

2. Mikes, V., Blein, J.-P., Milat, M.-L., Ponchet, M., Ricci, P., 1998. Use of elicitins as lipid carriers 

and their medical use. Patent PCT Int. Appl. 31 pp Coden; PIXXD2. 

3. Capasso, R., Cristinzio, G., Evidente, A.,Visca, C., Ferranti, P., Del Vecchio Blanco, F., Parente, A., 

Elicitin 172 from an isolate of Phytophthora nicotianae, pathogenic to tomato. Phytochem. 50, 

703-709. 

4. Capasso, R., Cristinzio, G., Di Maro, A. Ferranti, P., Parente, A., 2001. Syringicin a new α-elicitin from 

an isolate of Phytophthora syringae, pathogenic to citrus fruit. Phytochem. 58, 257-262. 


158

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