Breeding for resistance: breeding for Plum pox virus resistant apricots (Prunus armeniaca L.) in Spain

Plum pox virus , Dideron type (PPV-D), was first detected in Spain in 1984. Since then, it has spread among Japanese plum trees and apricot trees has been extremely rapid in the main producing areas. In Spain, breeding for resistance was the only efficient method for controlling the disease on apricot. Two breeding programmes are currently producing new hybrids resistant to the disease. The main problem encountered by both programmes is the difficult procedure needed for screening the trait that delays the programmes. Nevertheless, more than 8000 seedlings have been produced, two new varieties have been released and several advanced selections are under study. The procedure for screening sharka resistance has varied by techniques and cultivars used which has resulted in different hypotheses about inheritance of the trait. Additionally, several studies on mapping and molecular markers in progress could provide markers for molecular assisted selection that can increase the breeding efficiency.     

Breeding for resistance to plum pox potyvirus in the Czech Republic1

Breeding for plum cultivars resistant to plum pox potyvirus (PPV) is in progress in the Czech Republic, while projects for apricot and peach have started in 1991. The aim is to create an assortment of apricot and peach cultivars for northern regions of Europe in which PPV is widely distributed.     

Sensitivity of the detection of plum pox potyvirus by molecular assays1

Recently, plum pox potyvirus (PPV) has been found in Basilicata, southern Italy, on plum, apricot and peach. In 1992-09, we started a large-scale survey to verify the effectiveness of diagnostic methods used during seasons when it is difficult to reveal any presence of the virus. The assays were carried out by dot-blot hybridization on stone-fruit cultivars normally planted in this area. The virus was found, by dot-blot hybridization, to be present in seven cultivars of peach, four of apricot and one of plum. All plants were 8–10 years old and, except for two apricot cultivars, were not displaying any apparent symptoms in spring 1992. Five peach cultivars, intended for use as primary sources of propagation material, were then selected for further study, and assayed in 1992–11 by ELISA and RT-PCR. ELISA tests on these selected peach cultivars were consistently negative, while PCR tests were consistently positive. However ELISA tests gave positive results when repeated in 1993–05. These results not only suggest that primary propagation material should be tested by techniques more sensitive than ELISA, but also question the usefulness of carrying out tests during any phenological phases of the plant.     

Peach red marbling and peach sooty ringspot, two new virus-like degenerative diseases of Prunus

More than 600 Prunus samples were examined by using a nonradioactive digoxigenin-labelled RNA probe specific for hop stunt viroid (HSVd). Prunus salicina and Prunus armeniaca appeared to be better hosts than Prunus persica . The weak viroid concentration in flowers and young leaves of peach trees growing in the field did not permit its detection in such samples. The diagnosis was more reliable (about 85%) with bark and leaves aged 4 months and more, from regrowths of GF 305 peach seedlings inoculated and kept in the greenhouse. Detection of HSVd in leaves and bark of apricot and Japanese plum plants aged 3 months or more also proved reliable (about 80% and 90%, respectively). HSVd could be transmitted in apricot, peach and plum nucleic acid preparations to GF 305 peach seedlings by repeated stem slashing, and to cherries ( Prunus avium and Prunus serrulata ) by approach grafting with an infected P. salicina source. The viroid was eliminated from 18% of the clones obtained after thermotherapy.
In the course of this study, 25 selected Prunus accessions suspected to be infected by unusual diseases were analysed by hybridization with a HSVd-specific probe and by indexing on GF 305 peach seedlings in the greenhouse. Fifteen of these accessions were found to be infected by HSVd, 19 induced reddish marbling, and four induced small blackish spots on the leaves aged about 4 months. Repeated assays showed that these foliar symptoms were not caused by the viroid. Peach red marbling (PRMa) has not been associated with any known virus and seems to be caused by an infectious agent not yet described. That could also be the case with the agent of peach sooty ringspot (PSRS). PRMa and PSRS symptoms were reproduced by grafting and indexing, and their causal agents eliminated by thermotherapy in a significant fraction of the treated plants. They behave like viral agents and can infect the different Prunus species studied.     

Evaluation of apricot (Prunus armeniaca L.) resistance to Apple chlorotic leaf spot virus in controlled greenhouse conditions

Apple chlorotic leaf spot virus (ACLSV) seems to be the causal agent of apricot viruela disease. This disease has become an important problem for apricot production in Spain, mainly affecting the ‘Búlida’ cultivar, although no information is available about the behaviour of other cultivars with regards to ACLSV. In this study, the behaviour of 29 apricot cultivars against ACLSV (Apr 62 isolate) was evaluated under controlled conditions in an insect-proof greenhouse. Three different rootstocks, ‘GF305’ peach, ‘Real Fino’ apricot and ‘Adesoto’ plum, were first inoculated by grafting ACLSV-infected bark and were later grafted with the apricot cultivar to be evaluated. Apricot cultivars were evaluated during three cycles of study. ACLSV was asymptomatic on the leaves of all cultivars and rootstocks, so level of susceptibility or resistance was determined by virus detection through RT-PCR. ‘GF305’ rootstock showed a greater susceptibility level than ‘Real Fino’ and ‘Adesoto’. Most of the cultivars were susceptible to ACLSV with different levels of susceptibility, and only ‘Bergeron’ and ‘Mauricio’ were resistant.     

Epidemiology of Plum pox virus strain M in Greece

Plum pox virus has been endemic in Greece since 1967 causing important losses in apricot and to a lesser extent in peach crops. A survey undertaken in 1992 in public and private mother-tree plantations to estimate its incidence revealed that the virus was absent in isolated areas far from commercial stone-fruit crops. Virus titers decrease significantly during the hot months in the infected trees but re-increase in October–November permitting reliable detection. It is virulent M-type isolates which are effectively transmitted by aphids that are mostly recovered. Aphis gossypii and Hyalopterus pruni were the most abundant virus vectors captured during the small scale monitoring undertaken in apricot orchards in 1999 and 2000. Virus spread was monitored in two apricot orchards from 1996 to 2000 and analysed. Initial infections followed a completely random spatial pattern, while loose clusters appeared in succeeding years, to finally reach a uniform distribution representing high infection levels. The nearby ecological conditions greatly affected the rate of disease development.     

Breeding for resistance: conventional breeding for Plum pox virus resistant apricots (Prunus armeniaca L.) in Greece

A large apricot breeding programme has been conducted at NAGREF-Pomology Institute, Naoussa-Greece, for the control of sharka disease, since 1989. Ten apricot cultivars of North American origin: 'Stark Early Orange', 'Stella', 'NJA2', 'Sunglo', 'Veecot', 'Harlayne', 'Henderson', 'Goldrich', 'Orangered' and 'Early Blush', selected for their resistance to the highly virulent local strain of Plum pox virus (PPV)-M (Marcus), have been used as parents in crosses with quality cultivars, mainly with the local cv. Bebecou, from 1989 to 2003. Approximately 7000 hybrids have been created. Resistance to PPV was the main criterion of selection. Most hybrids have been subjected to artificial inoculation by PPV-M and examined for symptom expression for more than five years. Indexing to GF-305, as well as laboratory diagnostic tests, have been applied. The genetic analysis showed that: (1) 50% of the hybrids inherited resistance to PPV in the families where cvs. Stark Early Orange, NJA2, Sunglo, Veecot and Harlayne were used as a parent, and (2) 100% of the hybrids inherited resistance to PPV in the families where cv. Stella was one of the parents. Resistance to PPV appears to be under simple genetic control involving one gene locus. Promising apricot selections resistant to PPV-M have been released.     

Resistance to plum pox potyvirus in apricots1

The susceptibility of 115 apricot cultivars to plum pox potyvirus (PPV) has been examined, since 1981, in the experimental plots of the Pomology Institute at Naoussa and Skydra, Makedonia (GR). Inoculation was assured by aphids, transmitting strain PPV-M (Marcus) from naturally infected trees in adjacent peach orchards. For each cultivar, four to six trees were examined for at least 4 years. Observations on symptoms were made on leaves early in May and on fruits at maturity. Most cultivars expressed severe disease symptoms. Those without symptoms were inoculated by grafting onto heavily infected old apricot trees. The grafted shoots were tested for PPV in the following year by ELISA and on the woody indicator GF305. The cultivars which were rated as resistant after artificial inoculation and ELISA came from North America: Early Orange, Stella, NJA2, Sunglo, Veecot, Harlayne, Goldrich and Henderson. Most of these have been crossed with quality cultivars for the creation of resistant hybrids. The PPV resistance of large numbers of these apricot hybrids is now under investigation.     

History and importance of plum pox in stone-fruit production1

Plum pox potyvirus (PPV), which first appeared in Bulgaria in 1915, has spread to nearly every European country, and can today also be found in various eastern Mediterranean countries. The pest is of great economic importance especially in some southern and central European countries, causing considerable losses in production of primarily plum, apricot and peach. The extent of losses varies between countries of different climates, depending on cultivars and virus strains. In joint infections with other viruses, PPV provokes synergistic effects, enhancing the economic importance of the pest. Since PPV is transmitted by insects, spread of the virus can be reduced only by eradicating sources of infection and use of virusfree propagating material. Thus introduction of more reliable detection techniques is essential, facilitating mass testing of trees. Close collaboration in studying strain-related problems of PPV and in searching for resistant and tolerant cultivars is similarly important.     

Control and monitoring: monitoring and eradication of sharka in south-east Italy over 15 years

When the first foci of sharka were discovered in Puglia region (south-east Italy) in the late 1980s, the regional agricultural authorities launched a programme for Plum pox virus (PPV) monitoring and disease eradication. The infecting virus strain was identified as PPV-D. From 1989 to 1993, a strong eradication campaign was successfully carried out involving 13 plum and 2 apricot orchards with different levels of infection. During 1994–2000, besides plum, apricot and peach, monitoring was extended to sweet cherry. At that time, surveys and testing did not reveal any new PPV focus, but the eradication of infected trees continued in a couple of orchards. In 2001–05, particular attention was paid to peach, as devastating PPV-M outbreaks had developed in other areas of the country. A new PPV focus was found in apricot, caused by PPV-Rec, which was promptly eradicated. In the following two years, surveys in the once infected orchard and surrounding peach plantings did not detect any virus spread. The endeavour has taken 15 years making this PPV monitoring and eradication programme the longest in Italy. Its overall results indicate that the fruit tree industry in Puglia region can now be regarded as essentially PPV-free.     

Approaches to pathogen-mediated resistance breeding against plum pox potyvirus in stone-fruit trees1

In a programme for developing systems which allow the transfer of foreign genes into apricot cultivars, we have tested cotyledons of immature embryos, somatic embryos and leaf discs. Apricot plants have been transformed, and then regenerated, with Agrobacterium tumefaciens strain LBA 4404 containing various binary plasmids: pBinGUSint, carrying the marker gene β-glucuronidase (GUS), and pBinPPVm, carrying the coat-protein gene of plum pox potyvirus (PPV). The marker gene GUS was used for visual evaluation of the efficiency of the transformation system. The coat-protein gene was used in the hope of introducing coat protein-mediated resistance to one of the most important stone-fruit pathogens in Europe and the Mediterranean area.     

Epidemiology of sharka disease in Spain

PPV was first detected in Spain in 1984 in Japanese plum ( Prunus salicina Lindl) cv. Red Beaut and spread very quickly to other Japanese and European plums and apricot cultivars but left peach cultivars unaffected. In the years following the detection of PPV, the predominant aphid species visiting Prunus orchards in Mediterranean areas were Aphis gossypii followed by Aphis spiraecola , the latter being the main aphid species found at present. Both species are considered to be the main vectors of PPV in Spanish early Prunus growing areas. Spatial analysis of the spread of PPV-D in Japanese plum and apricot trees confirmed the lack of significant association between immediately adjacent trees. The observed spatial pattern of sharka suggests a lack of movement of PPV-viruliferous aphid vectors to immediately adjacent trees and indicates their preferential movement to trees several tree spaces away. PPV-D is the only type currently present in Spain, with the exception of a PPV-M outbreak that was detected in and successfully eradicated from Aragón in 2002. The short-distance spread of PPV-M infection occurred as far as 12 m along the rows of peach trees. However, PPV-D has not been observed to spread through peach cultivars, despite being grown in the vicinity of heavily infected plots of apricot or Japanese plum trees.     

Untersuchungen zur Europäischen Steinobstvergilbung (ESFY) in Deutschland

From 2003 to 2007 surveys have been conducted in different stone fruit growing regions in southwest Germany to detect European stone fruit yellows (ESFY) disease in Germany. Samplings have been done regularly in selected reference orchards in the regions Neuwieder Becken, Rheinhessen, Vorderpfalz and Südpfalz in summer on trees showing ESFY typical symptoms as well as on branches of trees with unspecific symptoms. All samples have been analysed by PCR for infection with Candidatus Phytoplasma prunorum. The phytoplasma could be detected in all investigated regions on the cultivated Prunus species P. armeniaca, P. persica and P. domestica. No infection was found in wild Prunus species. The main spread of the disease appeared on apricot while peach and European plum were less affected. A good correlation between symptoms and molecular detection of the pathogen could be shown for the typical symptoms in summer and winter for apricot as well as for peach. During regular psyllid captures in the reference orchards the population dynamics of Cacopsylla pruni could be described in southwest Germany for several years. By PCR-testing all collected insects individually a yearly natural infection rate of about 1–2% of all individuals of C. pruni could be calculated.     

Serological characterization of Italian isolates of plum pox potyvirus1

An Italian isolate of plum pox potyvirus (PPV) from apricot, Ispave 17, was used as antigen for production of monoclonal antibodies. Six clones secreting specific antibodies to PPV were obtained. All these monoclonal antibodies were used to test a collection of different Italian PPV isolates, collected from plum, apricot and peach orchards, and other European isolates (including PPV-D and PPV-M serotypes), using DAS-ELISA, SDS-PAGE, western blot and GIEM. In western blot analysis, the PPV-M and PPV-D coat protein, detected directly from crude peach GF305 extracts, showed different electrophoretic mobility, the coat protein of PPV-M being slightly larger than that of PPV-D. ELISA tests, performed with fixed dilutions of antibodies and limiting dilutions of clarified samples, showed with some monoclonal antibodies a marked difference between PPV-M and PPV-D strains, at ratios greater than 1:40 (w/v). Also in GIEM some monoclonal antibodies gave a good labelling reaction only with PPV-D serotype. With the help of this differentiation, it was found that all Italian isolates tested were of the D serotype and none of the severe M strain of PPV, which has not been reported in Italy.     

Detection and characterization of phytoplasmas in diseased stone fruits and pear by PCR-RFLP analysis in Turkey

During the late summer-early autumn of 2002, surveys were carried out in Turkey to determine the presence of phytoplasma diseases in fruit trees. Phytoplasmas were detected and characterized by PCR-RFLP analysis and TEM technique in stone fruit and pear trees in the eastern Mediterranean region of the country. Six out of 24 samples, including almond, apricot, peach, pear and plum, gave positive results in PCR assays. RFLP analysis usingSspI andBsaAI enzymes of PCR products obtained with primer pair f01/r01 enabled identification of the phytoplasmas involved in the diseases. Stone fruit trees, including a local apricot variety (‘Sakıt’) and a pear sample, were found to be infected with European stone fruit yellows (ESFY, 16SrX-B) and pear decline (PD, 16SrX-C) phytoplasmas, respectively. This is the first report in Turkey of PD phytoplasma infecting pear and of ESFY phytoplasma infecting almond, apricot, myrobalan plum and peach; ESFY phytoplasma infecting Japanese plum was previously reported. http://www.phytoparasitica.org posting July 21, 2005.     

Detection of prunus viruses by print-capture PCR 1

A very sensitive and simple procedure has been developed for the preparation of plant samples before PCR amplification using direct tissue printing. This technique was successfully used for the detection of plum pox potyvirus, apple chlorotic leaf spot trichovirus, prunus necrotic ringspot ilarvirus and apple mosaic ilarvirus, in different stone-fruit material (peach, apricot, plum and almond). The results demonstrate the general applicability of this technique.     

Species, Cultivars, Rootstocks and Environment

World apricot production by no means meets the total need of the international market (fresh and processed fruits) and is far below the potential in view of the existing favourable geographic, climatic and agrotechnical conditions. The cause for this insufficient world production of apricots, particularly in certain countries which have favourable conditions for apricot culture and its expansion, should be investigated using scientific and technical concepts concerning the apricot decline syndrome (apoplexy, sudden wilting, withering of apricots). After the mutual assistance of research workers from different disciplines in investigating this complex disease, the economic security of apricot growing can be improved and apricot decline reduced to an acceptable level. Special attention must be paid to selecting appropriate cultivars, rootstocks or interstocks, locations and cultivation techniques.