Apple proliferation resistance in apomictic rootstocks and its relationship to phytoplasma concentration and simple sequence repeat genotypes

In an effort to select and characterize apple rootstock resistant to apple proliferation (AP), progenies from seven apomictic rootstock selections and their parental apomictic species, Malus sieboldii and M. sargentii, were compared to standard stocks M 9 and M 11. Seedlings derived from open pollinated mother plants were grafted with cv. Golden Delicious and grown under natural infection conditions. The progenies differed greatly in resistance to the AP agent 'Candidatus Phytoplasma mali'. Progenies of M. sieboldii and its descendent rootstock selections D2212, 4608, 4551, and D1131 showed a high level of resistance, whereas progenies of M. sargentii and its descendent selections D1111 and C1828 proved susceptible. M 9 and M 11 showed an intermediate level of resistance. Phytoplasma titer in roots of the M. sieboldii and M. sargentii progeny groups was similarly low, whereas the concentration in the standard stocks was 100 to 5,000 times higher. In trees on most of the resistant stocks, only a minority was colonized in the scion, while in trees on susceptible and standard stocks, infection rate was often higher. Also, the titer in the top of trees on resistant stocks was usually lower than in trees on susceptible and standard stocks. Four progenies derived from open pollinated M. sieboldii and M. sieboldii descendents were subjected to DNA typing using simple sequence repeat (SSR) markers. This study revealed that the selected groups consisted mainly of mother-like plants (apomicts) and type I hybrids (unreduced mother genotype plus one male allele at each locus). Type II hybrids (full recombinants) and autopollinated offspring were rare. In the 4608 progeny, trees grown on type I hybrid rootstocks were significantly less affected than trees on mother-like stocks. In other progenies with fewer or no type I hybrids, trees on type II hybrids and autopollinated offspring suffered considerably more from disease than trees on mother-like stocks.     

In vitro screening for resistance to apple proliferation in Malus species

A screening system for apple proliferation resistance was developed, based on in vitro graft‐inoculation with the causal agent ‘Candidatus Phytoplasma mali’. For this, in vitro cultures of the field‐resistant apomictic genotypes Malus sieboldii, H0909, D2212 and the susceptible Malus × domestica genotypes Golden Delicious and rootstock M9 were established, as well as in vitro cultures of Rubinette and Golden Delicious infected with ‘Ca. P. mali’ strains PM4 and PM6, respectively. Healthy in vitro shoots were inoculated by micrografting with infected shoots used as graft tip. After 6 weeks graft contact no significant differences for graft quality were observed between healthy and infected grafts. Mortality of grafts and transmission rates were not significantly different among the different genotypes. The phytoplasma concentration in inoculated shoots was determined at different times post‐inoculation (p.i.) by quantitative real‐time PCR. Infected M. sieboldii and D2212 had lower phytoplasma concentration than the susceptible controls and showed no symptoms. H0909 showed an intermediate behaviour exhibiting lower phytoplasma concentrations with strain PM4 but growth was affected. The dynamics of phytoplasma concentration reached a maximum at 6–8 months p.i. for all genotypes but the values for 3–5 and 10–12 months p.i. were similar. The resistance of M. sieboldii and D2212 was confirmed in vitro. A significant difference in phytoplasma concentration was observed between strains PM4 and PM6.     

Use of SSR markers to assess sexual vs. apomictic origin and ploidy level of breeding progeny derived from crosses of apple proliferation-resistant Malus sieboldii and its hybrids with Malus × domestica cultivars

To obtain apple rootstocks resistant to apple proliferation and suitable to modern fruit growing, 24 cross-combinations were performed over a 5-year period using Malus sieboldii and its hybrids as donors of the resistance trait and standard apple rootstock Malus  ×  domestica genotypes as donors of agronomic value. Breeding with these genotypes was achieved despite different degrees of apomixis and polyploidy. Sets of five to six locus-specific microsatellite markers were identified for characterizing each progeny. Supported by flow cytometry these markers were applied to infer mode of reproduction, genomic constitution and ploidy level. Microsatellites allele composition identical to the maternal parent was revealed in 1668 of 3032 seedlings indicating seed formation through apomixis. Complete genetic recombination was found in 398 seedlings. The remaining hybrids displayed a higher ploidy than that of the parental plants which was consistent with the fertilization of unreduced egg cells. Thus, for each cross-combination, microsatellite loci were identified which enabled a reliable prediction of the ploidy level. They can now be applied in routine screening to distinguish sexual from apomictic progeny.     

Phytoplasma transmission by in vitro graft inoculation as a basis for a preliminary screening method for resistance in fruit trees

In vitro grafting was tested as a technique for inoculating Prunus rootstock Prunus marianna GF 8-1 with European stone fruit yellows (ESFY) phytoplasmas and apple rootstock Malus pumila MM106 with apple proliferation (AP) phytoplasmas. In vitro shoot cultures of ESFY-infected Prunus marianna GF 8-1 and AP-infected Malus pumila MM106 were used as graft inoculum to transmit the phytoplasmas to the respective healthy rootstock. Phytoplasma transmission was assessed after a graft contact of 1, 2 or 3 months. Healthy autografts were used as controls to monitor parameters of in vitro grafting. Successful graft union formation ranged from 58 to 79% irrespective of the plant species and the sanitary state of the graft. Pathogen-specific polymerase chain reaction (PCR) was used to test the inoculated rootstocks for the presence of ESFY and AP phytoplasmas, respectively. The rate of ESFY phytoplasma transmission in successful Prunus -grafts increased from 69 to 94% with the time of contact. AP phytoplasma transmission to Malus occurred in 80 to 97% of successful grafts. To our knowledge this is the first report of phytoplasma transmission by grafting in vitro . The results provide a good basis for the establishment of a preliminary in vitro screening method for phytoplasma resistance in Prunus and Malus .     

Detection and Indentification of European Stone Fruit Yellows and Other Phytoplasmas in Wild Plants in the Surroundings of Apricot Chlorotic Leaf Roll-affected Orchards in Southern France

Between 1994 and 1998 a field study was conducted to identify plant hosts of the European stone fruit yellows (ESFY) phytoplasma in two apricot growing regions in southern and southwestern France where the incidence of apricot chlorotic leaf roll was high. A total of 431 samples from 51 different plant species were tested for the presence of phytoplasmas by PCR using universal and ESFY-specific primers. ESFY phytoplasma was detected in six different wild growing Prunus species exhibiting typical ESFY symptoms as well as in symptomless dog rose bushes (Rosa canina), ash trees (Fraxinus excelsior) and a declining hackberry (Celtis australis). The possible role of these plant species in the spread of ESFY phytoplasma is discussed. PCR-RFLP analysis of ribosomal DNA amplified with the universal primers was carried out to characterize the other phytoplasmas found. Thus, elm yellows phytoplasma, alder yellows phytoplasma and rubus stunt phytoplasma were detected in declining European field elm trees (Ulmus carpinifolia Gled), in declining European alder trees (Alnus glutinosa) and in proliferating Rubus spp. respectively. The presence of rubus stunt phytoplasma in great mallow (Malva sylvestris) and dog rose was demonstrated for the first time. Furthermore, the stolbur phytoplasma was detected in proliferating field bindweed (Convolvulus arvensis) and a previously undescribed phytoplasma type was detected in red dogwood (Cornus sanguinea). According to the 16S rDNA-RFLP pattern this new phytoplasma belongs to the stolbur phytoplasmas group.     

Mapping the spread of apricot chlorotic leaf roll (ACLR) in southern France and implication of Cacopsylla pruni as a vector of European stone fruit yellows (ESFY) phytoplasmas

An epidemiological study on European stone fruit yellows (ESFY) phytoplasmas infecting Prunus fruit trees was carried out from 1994 to 2000 in Languedoc-Roussillon (southern France). The spread of the disease was monitored for 7 years by visual observation of symptoms and by PCR detection of the phytoplasma in an experimental orchard planted with apricot hybrid seedlings. This indicated that aerial vectors were responsible for disease spread, and that transmission rates were low at the beginning of the spread. Seventy thousand homopteran insects were captured within and in the surroundings of highly ESFY-infected apricot orchards, of which about 10 000 were used in PCR and nested-PCR assays with universal ribosomal and ESFY-specific nonribosomal primers to detect ESFY phytoplasmas. The other insects were confined in cages for trials of transmission to test plants. ESFY phytoplasmas could not be detected by PCR in any of the leafhopper species captured but could be detected in the psyllid Cacopsylla pruni caught on Prunus domestica and Prunus cerasifera rootstock suckers of apricot trees and on Prunus spinosa . Nested PCR revealed ESFY phytoplasmas in one individual of the deltocephalid Synophropsis lauri captured on an apricot tree. Transmission trials confirmed the role of Cacopsylla pruni as the ESFY phytoplasma vector in France. When apricot seedlings were used as bait plants from April to November during two consecutive years, no natural transmission could be demonstrated. However, one out of 50 apricot seedlings left for the whole year in the orchard became infected. An early spring ESFY infection is in agreement with both the natural transmission results and the life cycle of Cacopsylla pruni .     

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.     

Pcr Assay for specific detection of European stone fruit yellows phytoplasmas and its use for epidemiological studies in France

DNA amplification by polymerase chain reaction was used to specifically detect phytoplasmas associated with severe decline diseases of European stone fruits. PCR primers were designed according to the partial sequence of a nonribosomal genomic fragment of European stone fruit yellows phytoplasmas obtained by direct sequencing of a specific PCR product. A PCR assay was developed which resulted in specific amplification of a 237 bp-DNA fragment from total DNA extracts derived from over 300 stone fruit samples. No PCR product was obtained with DNA from healthy controls or plants diseased with various other phytoplasmas, e.g. the closely related apple proliferation and pear decline phytoplasmas. Phytoplasma infection was checked in all samples by PCR amplification with universal ribosomal primers. Detection rate with specific and universal primers was correlated by 97%. European stone fruit yellows phytoplasmas were detected in samples of 114 out of 139 examined orchards which represent the major stone fruit growing regions of France. Typical symptoms like chlorotic leaf roll in summer and off-season growth in winter were correlated by 95% to the presence of phytoplasmas. However, phytoplasmas were also detected in 51% of samples derived from trees showing non-specific symptoms. A comparison study including 201 samples showed that 81% of the PCR-positive samples were also tested positive using fluorescence microscopy with DAPI staining.     

Inheritance of apple proliferation resistance by parental lines of apomictic <Emphasis Type="Italic">Malus sieboldii</Emphasis> as donor of resistance in rootstock breeding

To study inheritance of Malus sieboldii-derived apple proliferation resistance, 14 cross combinations were performed with the tetraploid apomictic M. sieboldii and first and second generation parental lines as donor of resistance and Malus x domestica scion cultivars and apple rootstocks as donor of pomological traits. In the progeny examined mainly three classes were present consisting of mother-like plants with the allele composition of the maternal apomict (ML), hybrids based on fertilization of an unreduced egg cell (hybrid I), and fully recombinant plants (hybrid II). Two-year screening of inoculated plants in the nursery revealed that progeny classes ML and H I responded similarly to infection and that about half of the progeny showed satisfactory resistance. No appropriate resistance was identified in progeny class H II. This might be due to the fact that in fully recombinant offspring M. sieboldii haplotypes have been reduced from 4n to 1-2n or were entirely lost. Following nursery-growing, promising trees were evaluated for six more years in the orchard. Nearly all of them showed satisfactory resistance but were mostly less productive and more vigorous than trees on clonal standard rootstock M9. However, mainly among the offspring of progeny 4608 × M9, resistant genotypes were identified showing pomological properties similar to M9.