Interspecific transfer of resistance to Plum pox virus from almond to peach by grafting

Grafting almond variety ‘Garrigues’ onto ‘GF305’ peach seedlings heavily infected by Plum pox virus (PPV) progressively produces the disappearance of viral symptoms and drastically reduces virus accumulation in ‘GF305’ rootstock, in most cases to undetectable levels. This response appears to be specific between almond and peach, as it was not consistently observed by grafting ‘Garrigues’ onto other Prunus species such as plum (‘Adesoto’) or apricot (‘Real Fino’). The ability to induce resistance to PPV in ‘GF305’ was transmitted to the sexual descendants of Garrigues. Furthermore, grafting ‘Garrigues’ onto ‘GF305’ before PPV inoculation completely prevented virus infection, showing that the resistance is constitutive and not induced by the virus. This fact suggests that resistance may be due to the transfer of a defence factor from ‘Garrigues’ almond through the graft union and its interaction with specific factors of ‘GF305’ peach to produce the antiviral response. These results open new avenues to potential protection against PPV in peach, the most economically important species among stone fruits.     

Differences in Strain Virulence of the European Stone Fruit Yellows Phytoplasma and Susceptibility of Stone Fruit Trees on Various Rootstocks to this Pathogen

Twenty strains of the European stone fruit yellows (ESFY) phytoplasma showed great differences in virulence when examined by graft inoculation of trees on peach, peach hybrid GF 677 and P. 'Marianna' GF 8/1 rootstocks. The most virulent strains killed all trees on peach rootstocks whereas the mild strains did not cause mortality but induced only mild foliar symptoms and slightly reduced vigour. Virulence often depended on the pathogen–scion combination and was in several cases most severe when the scion consisted of the original host of the pathogen. To examine resistance in stone fruits, trees on a total of 23 rootstocks were inoculated with the ESFY strains. Trees on the Prunus domestica stocks Ackermann's, Brompton and P 1275 and on Prunus cerasifera stock Myrabi were little affected. Slightly more damage occurred in trees on rootstocks GF 677, GF 8–1, and the P. insititia stocks St Julien A and St Julien GF 655/2. Ishtara, P. cerasifera stock Myrobalan, and peach rootstocks Higama and GF 305 were shown to be moderately susceptible and a high susceptibility was found in trees on peach rootstocks Montclar, peach seedling, Rutgers Red Leaf, and Rubira, on apricot seedlings and St Julien 2. Of flowering cherry trees on various rootstocks, the least susceptible were those on Gisela 3 and F 12/1 whereas Gisela 1, Weihroot 158 and Gisela 5 were more affected. Phytoplasmas were detected by either DAPI (4'-6-diamidino-2-phenylindole) staining or polymerase chain reaction in all rootstocks and scions tested. However, detection frequency and phytoplasma concentrations were usually lower in the more tolerant hosts than in susceptible genotypes.     

Susceptibility of Prunus rootstocks to natural infection of Plum pox virus and effect of mineral oil treatments

The use of rootstocks that are less susceptible or resistant to natural Plum pox virus (PPV) infection and/or the application of mineral oil treatments are two possible strategies to reduce viral incidence in nursery plots. We evaluated the susceptibility of Prunus rootstocks used in the Spanish stone fruit industry and the effect of mineral oil treatment (Sunspray Ultrafine at 1%) on the spread of the virus at two different localities in Valencia, Spain, under different natural PPV inoculum pressures (high inoculum pressure, 2006–08; low inoculum pressure, 2006–07). Samples from both plots were analysed by double-antibody sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA) and spot real-time RT-PCR. Under high inoculum pressure, the assayed rootstocks exhibited significant differences in their susceptibility to natural infection. The most susceptible rootstocks at the end of the experiment were Adesoto 101 and Mariana GF8-1. Cadaman and Garnem rootstocks presented the fewest PPV-infected plants; these infections could be detected only by spot real-time RT-PCR. No differences among the assayed rootstocks were found under low PPV inoculum pressure. Aphid species were monitored using Moericke yellow water traps and sticky-plant methods at both localities in May 2006 and 2007. Aphis spiraecola was the most abundant aphid species monitored by both methods at both localities. The average percentage of A. spiraecola carrying PPV PCR-amplifiable targets was 30.37% in the plot with high PPV inoculum pressure and only 7.98% in the plot with low inoculum pressure. We found significant differences in PPV incidence between Mariana GF8-1 plots that were treated with mineral oil and those that were not treated after one year under natural conditions and high PPV inoculum pressure.     

Localisation and movement of Plum pox virus in apricot stem tissues

Localisation and movement of Plum pox virus (PPV), sharka disease, in stem tissues of susceptible and resistant apricot (Prunus armeniaca L.) cultivars was studied. Two different assays were performed. In the first assay, apricot cultivars were grafted on to a non‐inoculated GF305 peach rootstock and, after two months, the sprouted apricot was inoculated by chip‐grafting. In the second assay, apricot cultivars were grafted on to a previously chip‐inoculated GF305 showing strong PPV symptoms. Localisation of virus was studied in apricot stem by immuno‐tissue printing and sharka symptoms in GF305 and apricot leaves were also observed. Virus was mainly localised in the xylem, and sometimes in the cortex and pith. Results revealed that, while all the cultivars allowed limited virus movement from the inoculation point, only the susceptible cultivars (Screara, Bebeco and Colomer) allowed long distance movement and even showed symptoms in leaves.     

Evaluation of Resistance of Stone Fruit Rootstocks to Phytophthora Crown Rot*

The pathogenicity of Phytophthora citrophthora, Phy‐tophthora cactorum and Phytophthora megasperma strains was confirmed on 2‐year‐old stone fruit rootstocks artificially inoculated in the field. Phytophthora mega‐sperma was not virulent or less virulent than P. cactorum or P. citrophthora. The rootstocks Damas and AN 1/7 were resistant whereas the rootstocks Jl, Titan x Nemaguard 5/1, GF 677, Myrandier 613, Italian x Nemaguard 1 and Myrandier 617 were susceptible to P. citrophthora. The rootstocks Tsukuba 9, Tit‐an x Nemaguard 4/7, AN 1/2, AN 1/3, St. Mien 655/2 and Bl showed medium susceptibility to this species. The isolates of P. cactorum induced more extensive necrosis in Myrandier 617 and Italian x Nemaguard than in Myr‐andier 613, GF 677, Jl (ADAFUEL), Tsukuba 9 and St. Julien 655/2. Among seven rootstocks, Tsukuba 5 and St. Julien 655/2 were more resistant than Myrandier 613, Myrandier 617, Jl (ADAFUEL), Tsukuba 9 and St. Julien 655/2.     

Analogues of virus resistance genes map to QTLs for resistance to sharka disease in <Emphasis Type="Italic">Prunus davidiana</Emphasis>

Plum pox virus (PPV), the causative agent of sharka disease in Prunoideae, is one of the most serious problems affecting stone fruit production in Europe and America. Resistance to PPV was previously described in a Prunus davidiana clone, P1908, and introduced into peach (Prunus persica) genotypes. Genetic resistance to PPV displays a complex pattern of quantitative inheritance. An analysis of quantitative trait loci (QTLs) for resistance was performed on an F1 interspecific peach population obtained from a cross between the susceptible nectarine cultivar Summergrand and P. davidiana. The hybrids were graft-inoculated with PPV in duplicate following a classical procedure. The incidence of infection was evaluated four times, over two vegetative cycles, by symptom observation and enzyme-linked immunoadsorbent assays (ELISA). Restriction of systemic downward movement of the PPV virus was also evaluated by testing the susceptible rootstocks. Using both analysis of variance and non-parametric tests, six genomic regions involved in PPV resistance were detected. Depending on the scoring data considered, between 22 and 51% of the phenotypic variance could be explained by the quantitative model. One QTL, located in the distal region of linkage group 1, maps in a genomic region that is syntenic to the location of a resistance gene previously identified in the apricot cv. Goldrich. Some QTLs appeared to be temporally specific, reflecting the environmental dependence of PPV-resistance scoring. Candidate gene fragments were amplified by PCR, isolated and mapped on the peach interspecific linkage map. We report here the co-localization of three analogues of virus resistance genes with two distinct genomic regions linked to PPV resistance in P. davidiana.Electronic Supplementary Material Supplementary material is available for this article at     

Relative Resistance of Four Peach Rootstocks to Phytophthora cactorum and Phytophthora megasperma

Shoot samples of four peach rootstocks that are important to the peach industry in Greece: KID I, GF305, GF677, and PR204, were inoculated with Phytophthora cactorum and Phytophthora megasperma in the field and in the glasshouse and were then evaluated with regard to susceptibility. The pathogenicity of P. cactorum and P. megasperma to peach rootstocks was confirmed with an excised twig assay. The peach rootstocks showed differential susceptibility to P. cactorum . GF305 was the least susceptible and KID I was the most susceptible, which suggests that the latter rootstock is unsuitable for orchards in which the conditions are favourable for Phytophthora diseases. GF677 and PR204 were moderately susceptible. The plants that were inoculated with P. megasperma in the field and in the glasshouse showed no sign of infection whereas twigs inoculated in vitro with P. megasperma developed necrosis. GF305 was the most resistant, KID I was the most susceptible and PR204 and GF677 were moderately resistant. The present results demonstrate that none of the four peach rootstocks used in this study was completely resistant to P. cactorum , particularly when rootstocks were flooded periodically to enhance disease development. Therefore an integrated approach including host and resistance cultural practices is recommended to manage diseases caused by P. cactorum in peach orchards in Greece.     

Spread and competitiveness of Plum Pox Virus: Rec and ‐D strains in experimental plum orchard

Sharka disease caused by Plum pox virus (PPV) is endemic in Bulgaria. The speed and mode of spread of PPV‐Rec and PPV‐D isolates, molecularly characterised in advance and introduced in an experimental plum orchard to serve as permanent sources of infection was studied from 2008 to 2015. The trees were visually inspected for symptoms development and tested by DASI‐ELISA twice each year. The positive samples were serologically (‐M and ‐D specific MAbs) and molecularly (IC‐RT‐PCR) strain‐typed, and sequenced in (Cter)NIb‐(Nter)CP coding region. Artificially inoculated trees were found infected in the spring of 2009. After 7 years 43 trees (11.2%) became naturally infected. The majority of the trees were infected by PPV‐Rec (65%), while only one tree (2.5%) by PPV‐D. Nevertheless PPV‐M was not introduced in the orchard, this strain was found in single and mixed infections with PPV‐Rec strain. None of the isolates in naturally infected trees was identical to PPV‐Rec and PPV‐D inoculums, while several of them were a 100% identical to the respective PPV‐Rec, PPV‐D or PPV‐M isolates from neighbourhood‐infected trees. After the initial random pattern of PPV spread, aggregation of the new infections around the trees infected in previous years was observed. The presence of only Phorodon humuli and Hyalopterus pruni complex, known as PPV vectors and their poor abundance (5.0% from all caught aphids), together with some specific features of the landscape influenced the slow natural development of PPV in the experimental orchard.     

The plant growth‐promoting fungus Fusarium equiseti and the arbuscular mycorrhizal fungus Glomus mosseae stimulate plant growth and reduce severity of anthracnose and damping‐off diseases in cucumber (Cucumis sativus) seedlings

To alleviate the environmental contamination due to persistent chemical usage, approaches to integrated pest management were conceived. In this perspective, microbe–microbe interactions such as mycorrhizal relationships with other soil microbiota in the rhizosphere like the plant growth‐promoting fungi (PGPF) are particularly important. Better understanding of the interactions between beneficial microbial groups is imperative in the identification of possible synergistic or antagonistic effects to improve their practical usage as biocontrol agents or biofertilizers. In this study, the consequence of co‐inoculation of the arbuscular mycorrhizal fungus (AMF) Glomus mosseae (Gm) and the PGPF Fusarium equiseti (isolates GF18‐3 and GF19‐1) in terms of plant growth enhancement, root and rhizosphere colonisation, and development of anthracnose (Colletotrichum orbiculare) and damping‐off (Rhizoctonia solani AG‐4) diseases in cucumber plants was investigated under controlled conditions. The amendment of either GF18‐3 or GF19‐1 singly or in combination with Gm indicated a general tendency to significantly enhance the shoot dry weight (SDW) of cucumber plants at 4 weeks after planting (WAP). Similarly, Gm alone significantly enhanced SDW at 4 WAP. Gm showed a tendency to depress root colonisation by F. equiseti but such antagonistic effect was not observed in the rhizosphere soil. Both GF18‐3 and GF19‐1 significantly reduced percent root colonisation of Gm. However, these general tendencies may vary with the inoculum densities of AMF and PGPF. Both F. equiseti and Gm inoculated singly significantly increased percent of protection against anthracnose, but the combined inoculation was more effective in controlling the disease compared to single inoculation. The inoculation of the cucumber seedlings with GF18‐3, GF19‐1 or Gm, 6 or 12 days prior to damping‐off pathogen inoculation, increased percent of protection against damping‐off disease. This study shows that the co‐inoculation of F. equiseti and Gm resulted in additive effect on the suppression of anthracnose disease in cucumber.     

Effects of high grafting on tomato plants infected by Meloidogyne incognita and Ralstonia solanacearum

The root‐knot nematode Meloidogyne incognita is known to increase the severity of bacterial wilt in many solanaceous crops. In Japan, several bacterial wilt‐resistant rootstocks that have the M. incognita resistance (Mi) gene in their genome have been developed for tomatoes. In this study, we aimed to examine whether the presence of Mi gene‐breaking M. incognita population affects the development of bacterial wilt in bacterial‐wilt‐resistant tomato rootstocks with Mi in their genetic background. We also aimed to examine the possibility of using high‐grafted tomatoes to control bacterial wilt in plants infected by M. incognita. Our results indicate that the resistance to bacterial wilt was easy to break in usual‐grafted tomato plants infected with M. incognita and that M. incognita enhanced the vertical movement of Ralstonia solanacearum in the bacterial‐wilt‐resistant tomato rootstocks. In addition, our results suggest that high grafting led to significantly less wilting in the plants infected by M. incognita than did usual grafting.     

Effects of earthworms, nematodes, cultivations and host plants on Verticillium wilt of peach and cherry

Verticillium dahliae but not V. albo-atrum Berth was isolated from eight out of twenty-one stone fruit orchards surveyed for Verticillium wilt disease in western New York. Wilt incidence was related to the cultivation of tomato or legumes as previous or inter-crop with stone fruit trees. A limited cross species inoculation using isolates of V. albo-atrum and V. dahliae from woody and herbaceous plants showed that peach and cherry were susceptible to both species. The effect of V. dahliae on growth of cherry seedlings in the presence of Tylenchorynchus claytoni, Pratylenchus penetrans and Meloidogyne hapla was compared. P. penetrans and M. hapla produced more severe growth reduction than T. claytoni. The adverse effect of Verticillium on the growth of cherry seedlings was greater acting together with any one of the three nematodes than acting alone. V. dahliae was shown to be capable of passage through earthworms without loss of infectivity.     

Genetic transformation and regeneration of mature tissues of woody fruit plants bypassing the juvenile stage

Regeneration and transformation systems from mature plant material of woody fruit species have to be achieved as a necessary requirement for the introduction of useful genes into specific cultivars and the rapid evaluation of resulting horticultural traits. We report here, for the first time, a procedure for genetic transformation and regeneration of mature tissues of woody plants that overcomes the long juvenile periods and high heterozygosity that are characteristic of most of these species. An improved regeneration frequency from mature explants was obtained by invigoration of the plant material through grafting of mature buds on juvenile seedlings. Co-cultivation of the explants in feederplates after inoculation with Agrobacterium tumefaciens resulted in enhanced transformation frequencies. Furthermore, in vitro shoot-tip grafting of the regenerated mature shoots on seedling rootstocks provided a rapid and efficient system for plant production. Citrus is the most extensivel y grown fruit crop worldwide and sweet orange (Citrus sinensis L. Osbeck) accounts for approximately 70% of the Citrus total production. Mature transgenic sweet orange plants have been obtained, which flowered and bore fruit in 14 months     

Identification of simple sequence repeat markers tightly linked to plum pox virus resistance in apricot

Sharka disease, caused by the plum pox virus (PPV), is one of the major limiting factors for stone fruit production in Europe and America. Attempts to stop the disease through the eradication of infected trees have been unsuccessful. Introgression of PPV resistance for crop improvement is therefore the most important goal in Prunus breeding programs. Due to time- and labour-consuming protocols, phenotyping for sharka is still the major bottleneck in the breeding pipeline. In this context, screening of seedlings at early stages of development and marker-assisted selection (MAS) provide the best solution for enhancing breeding efficiency. In this study, we generated 42 simple sequence repeat (SSR) markers from the peach genome assembly v1.0 and an apricot bacterial artificial chromosome clone identified in the physical map of the PPV resistance locus previously defined in apricot. Using a linkage mapping approach, we found SSR markers tightly linked to PPV resistance trait in all our progenies. Three SSR markers, PGS1.21 PGS1.23 and PGS1.24, showed allelic variants associated with PPV resistance with no recombinants in the crosses analysed. These markers unambiguously discriminated resistant from susceptible accessions in different genetic backgrounds. The results presented here are the first successful application of their use in MAS for breeding resistance in Prunus species.     

Hypersensitive response, cell death and histochemical localisation of hydrogen peroxide in host and non-host seedlings infected with the downy mildew pathogen Sclerospora graminicola

Hypersensitive response, cell death and release of hydrogen peroxide as measures of host and non‐host defense mechanisms upon inoculation with the downy mildew pathogen Sclerospora graminicola were studied histochemically at the light microscopy level. The materials consisted of coleoptile tissues of the highly susceptible (cv. HB3), highly resistant (cv. IP18293) and induced resistant pearl millet host seedlings and non‐host sorghum (cv. SGMN10/8) and cotyledon of french bean (cv. S9). Resistance up to 80% protection against the downy mildew pathogen was induced in the highly susceptible HB3 cultivar of pearl millet by treating the seeds with 2% aqueous leaf extract of Datura metel for 3 h. Time course study with the pathogen inoculated highly resistant pearl millet cultivar revealed the appearance of hypersensitive response in 20% of seedlings as necrotic spots as early as 2 h after inoculation. In contrast, a similar reaction was observed in the highly susceptible pearl millet cultivar only 8 h after inoculation with the pathogen. In induced resistant seedlings, appearance of hypersensitive response was recorded 4 h after inoculation. Delayed hypersensitive response was observed in both the non‐host species at 10 h after inoculation. Hypersensitive response in the seedlings of the highly resistant pearl millet cultivar 24 h after inoculation showed 100% hypersensitive response, which was not observed in susceptible and non‐host species, although the induced resistant seedlings showed 90% hypersensitive response after that period of time. Cell death in the tissues of the test seedlings was also observed to change with time. Statistical analysis revealed that the tissues of highly resistant pearl millet seedlings required 2.9 h to attain 50% cell death. Tissues of induced resistant and highly susceptible pearl millet seedlings required 4.65 and 6.50 h respectively. In non‐hosts, 50% cell death was not recorded. Quantification of hydrogen peroxide in the tissue periplasmic spaces of the test seedlings revealed 2.94 h as the time required for 50% hydrogen peroxide accumulation in the tissues of highly resistant pearl millet seedlings. Tissues of induced resistant and highly susceptible pearl millet seedlings needed 3.76 and 5.5 h respectively. Fifty percent hydrogen peroxide localisation in non‐hosts could not be recorded. These results suggested the involvement of hydrogen peroxide, cell death and hypersensitive response in pearl millet host defense against S. graminicola.     

The potential of Lagenaria rootstock to confer resistance to the carmine spider mite, Tetranychus cinnabarinus (Acari: Tetranychidae) in Cucurbitaceae

Antibiosis and resistance of six Cucurbita and two Lagenaria accessions to the carmine spider mite, Tetranychus cinnabarinus Boisduval, were evaluated in the laboratory. Significant differences among accessions were observed three days after the inoculation of detached leaf discs. The Lagenaria accessions, Slawi and Sus, proved to be the most resistant to mites, with average populations of mite eggs, 87 and 95%, respectively less than that of the susceptible C. pepo accession, Orangetti. The Cucurbita accessions, Tace, Brava, Tetsukabuto, Phoenix and TZ-148 had mite egg totals 4, 9, 13, 26 and 40%, respectively, less than those of accession Orangetti. The Sus accession of Lagenaria was resistant to T. cinnabarinus from the four-leaf stage until fruit set in laboratory and field tests. Grafting the susceptible Brava onto Sus rootstock increased the resistance of the scion to the same level as that of non-grafted Sus. Grafting the susceptible Cucumis melo Noy Yizre'el on resistant or susceptible rootstocks of Cucurbita and Lagenaria accessions did not affect its susceptibility to T. cinnabarinus. The results indicate that resistance to T. cinnabarinus can be transferred by grafting from Lagenaria stocks to Cucurbita scions but not in the opposite direction.