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V. Savino1, B. Di Terlizzi2, A.M. D'Onghia2 Terlizzi2, A.M. D'Onghia2,
M. Digiaro2, O. Murolo3, L. Catalano4
and G.P. Martelli1
1Dipartimento di Protezione delle Piante,
Università degli Studi and Centro di Studio del CNR sui
Virus e le Virosi delle Colture Mediterranee, Bari, Italy;
2Istituto Agronomico Mediterraneo, Valenzano (Bari),
Italy;
3Osservatorio per le Malattie delle Piante, Regione
Puglia, Bari, Italy;
4Consorzio Vivaistico Pugliese, Bari, Italy
In Apulia the phytosanitary status of fruit tree
crops (stone fruits, grapevine, olives and citrus) is unsatisfactory
much the same as in other Italian regions, but it is improving
thanks to the adoption of preventive measures, such as the use
of "virus tested" budwood. To this effect, a regional
programme for the certification of propagating material produced
by local nurseries was established. Sanitary selection of native
and newly introduced cultivars for stone fruits, olive, grapevine
and citrus was carried out in the main horticultural areas of
Southern Italy by visual inspection, biological indexing, immunoenzymatic
tests (ELISA) and molecular techniques. For many cultivars for
which it was not possible to identify candidate mother plants
of a sanitary condition compatible with certification requirements,
heat ther compatible with certification requirements,
heat therapy and/or in vitro culture was applied whenever
necessary. The Center of Conservation for Premultiplication, operating
in the framework of the Apulian certification programme, has available:
(253) stone fruits, (22) citrus, (34) grapevine and (22) olive
virus-free cultivars and/or clones. This high quality and broad-spectrum
propagating material is delivered to local nurseries, giving Apulia
region a national leading role in the production of certified
propagating material. Morever, this regional experience is now
being introduced in different Mediterranean countries thanks to
the international cooperation established between Apulian and
Mediterranean Research Institutes. Governments of four Mediterranean
countries (Albania, Lebanon, Malta and Palestine) have shown interest
for the Apulian certification protocols which is being used as
basis for the implementation of national programmes.
Diseases induced by intracellular infectious agents (viruses, viroids, phytoplasmas) represent a major threat to fruit trees, and may constitute a limiting factor to their growing. The wide geographical distribution of these disorders derives both from the inefficacy against viruses of the methods commonly used for controlling plant pathogens and from dispersal operated by vectors and propagative material. Man itself is the major responsiblegative material. Man itself is the major responsible for the long distance dissemination of infectious diseases, through the uncontrolled propagation and trading of infected stocks. It is not by chance that the species with the worst sanitary conditions are those object of extensive manipulations in the nurseries.
Surveys carried out in Southern Italy (Grayaa et al, 1993; Choueiri et al., 1993; Digiaro et al., 1994; Di Terlizzi et al., 1994; Savino et al., 1995; D'onghia et al., 1998) and in several Mediterranean countries (Martelli, 1986; Martelli et al., 1992; Merkuri et al., 1994; Myrta et al., 1996; Haidar et al., 1996; Zeramdini et al., 1996; Jawhar et al., 1996; D'Onghia et al., 1998; Mahafoudhi et al, 1998; Al Kowni et al., 1998) have shown how deteriorated is the health condition of fruit trees in the area, and how difficult is to find plants of acceptable sanitary status in the field. No sanitary improvement of fruit tree crops would therefore be possible without a strict prevention policy.
The first example of international action addressing the problem of the sanitary improvement of vegetatively propagated crops, was EEC Directive 68/193, containing provisions for the production of grapevine materials free from a couple of virus diseases (fanleaf and leafroll). In Italy, this Directive was applied promptly (DPR 1164/6this Directive was applied promptly (DPR 1164/69), but more than 20 years had to elapse before comparable legislative actions were taken for other crops. Eventually, decrees were promulgated by the Ministry of Agriculture for the voluntary certification of stone fruits (DM of 31.12.92), olive (DM of 16.6.93), citrus and pome fruits (DM of 29.10.93), but their implementation is slow and far from being generalized.
This paper decribes in short how the certification issue is being
addressed in Apulia (South Eastern Italy).
In Apulia, all started with the sudden outbreak of sharka, a much feared and devastating disease caused by plum pox virus (PPV), a few scattered foci of which were discovered some ten years ago in the region (Di Terlizzi et al., 1988). Apulia was known to be free from PPV, thus its introduction caused tremendous concern among growers, and the urgent demand for virus-free material.
Faced with this emergency, and because of the lack of national legislation, the Apulian regional government, decided to proceed autonomously. A decree (DPGR n° 893 of 20.10.1989 ) for the compulsory control of PPV was promulgated with the aim of eradicating extant infection foci and preventing PPV re-introduction with infected propagating material. To this effect, it was determined that new plum, apricot and peach tree orchards had to be established with PPV-fpeach tree orchards had to be established with PPV-free nursery material, regardless of its origin (Murolo et al., 1990). By imposing the use of budwood from mother plants that had undergone laboratory testing for ascertaining their PPV-free condition, this decree set up de facto a form of sanitary certification of the propagating material of the stone fruit species in question.
The organization of the mandatory certification of these species and relative rootstocks produced by Apulian nurseries, was further defined by the Regional Council a couple of years later (Deliberation n° 2514 of 6.6.1991). This act ratified the protocols describing the characteristics of propagative material, the controls to be carried out, the certification procedures, and established the duties of the agencies dealing with the different phases of the certification process.
Consequent to the establishment of the National Service for the
voluntary certification of plant propagation material (DM n°
289 of 2.7.1991), the Apulian regional government applied (DGR
n°1119 of 17.5.1993) for participating in the National Service.
With the same act, the Regional Certification Service was re-organized,
certification was extended to a wider range of stone fruit viruses
and to other fruit crops, and the structures where the different
phases of the process had to be carried out were identified. Currently,
the Apulian nursery productions of Currently,
the Apulian nursery productions of stone fruits (apricot, plum,
peach, cherry, almond), citrus, and olive are certified for freedom
from all diseases included in national certification protocols.
Scientific institutions in the region (Department of Plant Protection and Institute of Pomology of the University of Bari, Center of the Italian National Research Council for the Study of Viruses and Virus Diseases of the Mediterranean Crops, and the Mediterranean Agronomic Institute) have undertaken an extensive programme for the pomological and sanitary improvement of the regional fruit tree industry.
Only registered selections free from the infectious agents included in national protocols are eligible for certification. Thus, when required, selected stocks undergo sanitation treatments which, according to the plant species, consist of heat therapy, meristem tip culture, or micrografting. Controls for the assessment of the sanitary status, either before or after sanitation treatments, are based on different types of assays: (i) biological (indexing on woody indicators for all species except for olive, and mechanical transmission to herbaceous hosts); (ii) serological (ELISA, using polyclonal antisera and/or monoclonal antibodies); (iii) molecular (radioactive or cold probes, PCR); (iv) electrophoretic (for viroids and dsRNAs).
The outectrophoretic (for viroids and dsRNAs).
The outcome of the work carried out so far can be summarized as
follows:
Grapevine: 34 different clones (14 of roostocks and 20
of wine grape varieties) have been registered and 17 more (10
of wine grapes and 7 of table grape varieties) will be submitted
to registration within the current year. All the clones are free
from fanleaf, leafroll, rugose wood, and fleck, thus conforming
to the requirements of the Committee for the Evaluation of Grapevine
Varieties of the Ministry of Agriculture, that are stricter than
those of EEC Directive 68/193, and were all sanitized by heat
treatment and/or meristem tip culture.
Stone fruits: two types of materials are produced, i.e.
"virus-tested" free from PPV, and "virus-free",
tested for the absence from PPV and the ilarviruses (PNRSV, PDV,
ApMV), nepoviruses (ArMV, CLRV, CRLV, MLRSV, RRV, SLRSV, TBRV,
TomRSV) and trichoviruses (ACLSV) known to infect these species,
plus peach latent mosaic viroid (PLMVd). Over 250 selections (62
almonds, 35 cherries, 52 apricots, 38 plums, 47 peach, and 19
roostocks) meeting the sanitary requirements of certification
protocols have been identified. Most of these selections are of
the "virus-free" category; some have undergone sanitation.
All selections are currently under pomological evaluation.
Citrus: 22 selections (6 sweet oranges, 6 clementines,/I> 22 selections (6 sweet oranges, 6 clementines,
4 lemons, 2 limes, and 4 miscellaneus species) are available.
All selections are free from tristeza (CTV) (virus-tested) or
free from CTV plus infectious variegation (CVV), psorosis-ringspot
(CPSaV), stubborn (Spiroplasma citri), exocortis (CEVd),
and cachexia-xyloporosis (CCVd) (virus-free). All selections were
sanitized by micrografting.
Olive: 22 selections of different oil and table varietes have been identified and recognized by the Regional Certification Service. These selections are free from all viruses and other pathogens (Pseudomonas syringae pv savastanoi and Verticillium dahliae) included in the certification protocol, as ascertained by biological and laboratory testing (absence of dsRNAs). More sensitive molecular assays for virus detection are being developed.
All the above selections represent nuclear stocks (primary sources)
and are currently grown in insect-proof screenhouses in the experimental
farm of the University of Bari, at Valenzano. Waiting for the
National Certification Service to be fully operative, the Valenzano
outfit functions temporarily as Conservation Center for Premultiplication
under the control of the Regional Certification Service.
By appointment of the Apulian regional government the pre-multiplication fase of the certification process, i.e., the prodion fase of the certification process, i.e., the production of propagating material of "basic" category for the establishment of mother plant plots of "certified" category, is entrusted to the Centro di Ricerca e Sperimentazione in Agricoltura "Basile Caramia" (CRSA) of Locorotondo. Premultiplication activities are performed by the CRSA in different localities of the region, taking into account the pedo-climatic requirements of each species. EU funds (FEOGA), as well as public (regional, provincial, and local agencies) and private (nurseries) funds, were used for the setting up of the infrastructures needed.
Premultiplication of grapevine is temporarily done on the Jonic coast of Apulia at Palagiano (TA), on a surface of about 0.5 ha hosting 11 roostock clones and 10 wine grape clones, all selected by the University of Bari (UBA) and registered by the Ministry of Agriculture. By the end of 1998, a new outfit will be operational at Cannole (LE), on the Adriatic coast of Apulia, with a surface of about 4 ha. It is expected that the premultiplication of all grapevine clones, including those whose registration is pending, will be carried out at Cannole.
Olive is premultiplied at Palagiano, where the 22 selections registered by the regional authorities are grown on a surface of 0.25 ha. A glasshouse with heated benches for rooting olive shoots under mist is operational on the premises of the premultiplicatierational on the premises of the premultiplication outfit.
Mother plants of citrus and stone fruits of "basic"
category are grown under insect-proof screenhouses. "Basic"
citrus mother plants consists of 22 UBA selections plus 8 virus-free
selections of Spanish origin (AVASA). All are temporarily grown
at Palagiano, but will be moved shortly to a neighbouring outfit
in Massafra (TA). Stone fruits are grown at Locorotondo in screenhouses
that, by the end of 1998, will cover a surface of 2,000 sqm.
Whereas grapevine mother plant plots destined for the production of certified material are mostly grown by single nurseries, the trend for other species is that of managing the multiplication phase in a collective form. For example, the Consorzio Vivaistico Pugliese (Co.Vi.P.) established in 1991 and comprising 16 associated nurseries, conducts the only stone fruit tree multiplication centre currently operating in the region. Over 160 different varieties of apricot, cherry, peach, almond, plum, and their rootstocks, totalling more than 6,000 trees, are grown at Mottola (TA) on a surface of about 7 ha. This centre has a potentiality for the yearly production of about four million buds and one million seeds, which largely covers normal regional needs.
As to citrus and olive, it was the CRSA itself that, with the financial assistance of Co.Vi.P., established "cer financial assistance of Co.Vi.P., established "certifed" foundation blocks totalling 600 and 520 trees, respectively. However, private nursery groups are now planting two olive tree mother plant orchards with a total surface of about 8 ha, and one of citrus, with a surface of about 1 ha.
As to grapevines, in Apulia there are 257.5 ha of roostock mother
plants, 80% of which of "certified" category, and 201.5
ha of scion mother plants, 37% of which of "certified"
category (source: Istituto Sperimentale per la Viticoltura, Turi
branch). It may be worth pointing out that the grapevine certification
is handled by a specific separate service acting under the control
of the Istituto Sperimentale per la Viticoltura of the Ministry
of Agriculture. To simplify the procedures, avoiding overlapping
of competences, and possible controversies, it would be desirable
that a single National Certification Service for all plant species
be established, under the supervision of the Ministry of Agriculture
(Martelli, 1995)
The certification of grapevines and stone fruits now represents a well-established practice in Apulia. Consistently with the national trend, grapevines shows a slow but steady growth of "certified" nursery production at the expense of materials of "standard" category, especially for rootstocks. In Apulia, the delay in the premultiplication of certifipulia, the delay in the premultiplication of certified clones of local cultivars has somewhat slowed down the process which, however, is expected to receive new impulse from the activity of the CRSA centre.
"Certified" stone fruits nursery productions have virtually replaced "standard" material. The number of nurseries joining the voluntary regional certification programme has grown from 9 in 1991 to 14 in 1998, with a consequent significant increase in the production of certified materials (Fig. 1 and 2).
As to citrus and olive, in 1997 the Regional Certification
Service has certified 13,000 virus-free and 45,000 virus-tested
citrus plants, and expects to certify some 17,500 virus-free olives
in 1988. By the turn of the century the yearly production of certified
olive will likely outnumber 100,000 units.
The positive experience of certification in Apulia was made possibile
by the concomitance of several factors:
(a) Awareness of the problem. The alarm caused by the ingress of sharka and fireblight, and the realization of how widespread virus diseases are in fruit tree crops, highlighted the dangers of improvisation in nursery practices that neglect elementary prevention norms for controlling dangerous pathogens and restraining their spread.
(b) Existence of a very active nursery sectord.
(b) Existence of a very active nursery sector. Nursery
industry is important in the economic and social environment of
the region. A number of young entrepreneurs, open to innovation
and sensitive to the messages from the scientific world, currently
operate in the sector. The capillary information campaign carried
out by regional technical and scientific bodies, convinced the
most advanced and innovative of them to support the initiative
taken and to invest substantially in it, also for promoting the
image of the Apulian nursery industry.
(c) Presence of reference scientifc institutions. Institutions
with a consolidated experience and competence in sanitary and
pomological selection are present in the region. These institutions
have carried out field selection, sanitized diseased stocks whenever
necessary, identified primary sources, provided scientific support
in all phases of the certification process, and favoured its implementation
by contributing infrastructures and laboratory support for sanitary
checks.
(d) Political will. Regional authorities, especially the
Assessorship to Agriculture, have paid great attention to the
problem, providing enthusiastic collaboration, and ensuring technical,
legislative, and financial support.
The availability of high quality propagating material of a wide
range of plant species, gives Apulia a national lead in the production
of certified fruit treenal lead in the production
of certified fruit trees. The "Apulian system", tested
by several years of experience, is now being introduced in Mediterranean
countries like Albania, Lebanon, Malta and Palestine, which have
shown interest for the certification protocols developed in the
region, in view of the implementation of their own national programmes.
Fig. 1. "PPV-tested" stone fruit plants
produced in Apulia in the last eight years
Fig. 2. "Virus-free" stone fruit plants
produced in Apulia in the last five years