Genome mapping of an apple scab,a powdery mildew and a woolly apple aphid resistance gene from open-pollinated Mildew Immune Selection

Apple is host to a wide range of pests and diseases, with several of these, such as apple scab, powdery mildew and woolly apple aphid, being major causes of damage in most areas around the world. Resistance breeding is an effective way of controlling pests and diseases, provided that the resistance is durable. As the gene pyramiding strategy for increasing durability requires a sufficient supply of resistance genes with different modes of action, the identification and mapping of new resistance genes is an ongoing process in breeding. In this paper, we describe the mapping of an apple scab, a powdery mildew and a woolly apple aphid gene from progeny of open-pollinated mildew immune selection. The scab resistance gene Rvi16 was identified in progeny 93.051 G07-098 and mapped to linkage group 3 of apple. The mildew and woolly aphid genes were identified in accession 93.051 G02-054. The woolly aphid resistance gene Er4 mapped to linkage group 7 to a region close to where previously the genes Sd1 and Sd2, for resistance to the rosy apple leaf-curling aphid, had been mapped. The mildew resistance gene Pl-m mapped to the same region on linkage group 11 where Pl2 had been mapped previously. Flanking markers useful for marker-assisted selection have been identified for each gene.     

Phenolic acid content – a criterion for selection of resistant apple cultivars against Podosphaera leucotricha (Ell. and Ev.) Salmon

Development of powdery mildew (Podosphaera leucotricha) on five popular cultivars of apple, viz., Scarlet Gala, Golden spur, Mollies Delicious, Red Fuzi and Red Chief was studied to determine incidence–severity relationship. The disease was confined primarily to the vegetative terminal shoots early in the season which also traversed later onto other leaves. Several biochemical changes occur in the trees due to fungal/microbial infection. We studied the qualitative/quantitative changes in phenolic acids in apple-powdery mildew pathosystems. Scarlet Gala and Red Chief are very rich in phenolic acids, and had shown resistances to the pathogen but those with low amount of phenolic acids, viz., Golden spur, Mollies Delicious, and Red Fuzi, were highly susceptible. Thus, the quantity of phenolic acids (secondary metabolites) has been taken as a biochemical parameter in screening apple cultivars for resistance/susceptibility against powdery mildew of apple.     

Identification of SCAR markers linked to <Emphasis Type="Italic">Pl-w</Emphasis> mildew resistance in apple

Resistance to powdery mildew is an important objective for cultivar improvement programmes of apple and several different major genes for resistance to mildew are available. Molecular markers linked to such key traits can be used to screen progenies for resistant individuals. A progeny derived from the crab apple 'White Angel' (the source of Pl-w) was screened for resistance to mildew for two seasons in the glasshouse and four seasons in the field. DNA bulks of resistant and susceptible seedlings were screened with 176 AFLP primer combinations. Seven AFLP markers were identified that differentiated the bulks, and two of these markers were developed into SCARs, EM M01 and EM M02, mapping at 4.6 and 6.4 recombination units from Pl-w.     

Evaluation of microcapsule trunk injections for the control of apple scab and powdery mildew

A 3‐year field trial was conducted using established apple cv. Crown Gold and English oak (Quercus robur L.) to assess the efficacy of eight fungicides applied via microcapsule trunk injection against the foliar pathogens apple scab (Venturia inaequalis) and powdery mildew (Phyllactinia sp). In both apple cv. Crown Gold and English oak, the fungicide myclobutanil was not taken up when microcapsules were inserted into the tree vascular system at the root flare. Disease severity in injected trees, excluding myclobutanil, was lower over the following two growing seasons compared to water‐injected controls indicating seven of the eight fungicides used in this study provided a significant degree of protection against scab and powdery mildew infection. A difference in the magnitude of pathogen control achieved was recorded between fungicides. Of the fungicides tested, penconazole, pyrifenox and carbendazim significantly reduced disease severity and significantly increased leaf chlorophyll (Fv/Fm) and SPAD values as a measure of tree vitality and chlorophyll content, respectively, in both apple cv. Crown Gold and English oak over two growing seasons after microcapsule injection. Based on the results of this investigation, it is suggested that these three fungicides be used in preference to thiabendazole, fosetyl‐aluminium, triadimefon and propiconazole for the control of apple scab and powdery mildew where outbreaks of these foliar pathogens are problematic.     

Temporal isolation explains host-related genetic differentiation in a group of widespread mycoparasitic fungi

Understanding the mechanisms responsible for divergence and specialization of pathogens on different hosts is of fundamental importance, especially in the context of the emergence of new diseases via host shifts. Temporal isolation has been reported in a few plants and parasites, but is probably one of the least studied speciation processes. We studied whether temporal isolation could be responsible for the maintenance of genetic differentiation among sympatric populations of Ampelomyces, widespread intracellular mycoparasites of powdery mildew fungi, themselves plant pathogens. The timing of transmission of Ampelomyces depends on the life cycles of the powdery mildew species they parasitize. Internal transcribed spacer sequences and microsatellite markers showed that Ampelomyces populations found in apple powdery mildew (Podosphaera leucotricha) were genetically highly differentiated from other Ampelomyces populations sampled from several other powdery mildew species across Europe, infecting plant hosts other than apple. While P. leucotricha starts its life cycle early in spring, and the main apple powdery mildew epidemics occur before summer, the fungal hosts of the other Ampelomyces cause epidemics mainly in summer and autumn. When two powdery mildew species were experimentally exposed to Ampelomyces strains naturally occurring in P. leucotricha in spring, and to strains naturally present in other mycohost species in autumn, cross‐infections always occurred. Thus, the host‐related genetic differentiation in Ampelomyces cannot be explained by narrow physiological specialization, because Ampelomyces were able to infect powdery mildew species they were unlikely to have encountered in nature, but instead appears to result from temporal isolation.     

Characterization of six polymorphic microsatellite loci from Ampelomyces quisqualis,intracellular mycoparasite and biocontrol agent of powdery mildew

Ampelomyces spp. are common intracellular mycoparasites of powdery mildews worldwide and a strain has been commercialized as a biocontrol agent against these plant pathogens. In light of recent genetic analyses revealing high internal transcribed spacer sequence variability among Ampelomyces strains on different host plant mildews, yet no sequence variability within the Malus strain, polymorphic microsatellites were required to permit biocontrol and ecological studies of the complex apple/apple mildew/Ampellomyces tritrophic interaction. For this purpose, described here are the primers to amplify six polymorphic microsatellite loci from Ampelomyces quisqualis isolated from mycelia of the apple powdery mildew fungus, Podosphaera leucotricha.     

Cytological techniques for use in apple breeding

Details are given of improved cytological squash techniques for mitotic and meiotic counts which are especially useful in apple breeding. The cytological characteristics of scion varieties (including triploids such as the recently named Suntan), apple rootstocks (including tetraploids), and second backcross progenies of the mildew resistant species Malus hupehensis (2n= 51) to the cultivated apple (in which most seedlings were found to be diploid) are discussed with reference to their implications for apple breeders.     

The effect of powdery mildew on the number of prey consumed by Typhlodromus pyri (Acari: Phytoseiidae)

Abstract:  Prey consumption by Typhlodromus pyri Scheuten was studied in the presence and absence of apple powdery mildew, Podosphaera leucotricha (Ell. and Everh.) under constant laboratory conditions. Eggs of Tetranychus urticae Koch were offered to predatory mites as a prey. Seven densities ranging from five to 100 T. urticae eggs per arena were used. Mildew conidia (approximately 0.5 mg) were added to half of the arenas by brushing them from infected apple leaves. A single adult female of T. pyri was introduced onto each arena and number of prey eggs consumed was counted 12 h later when the predator was offered new T. urticae eggs again and the procedure was repeated once. Data showed that predators consumed in both experimental periods nearly all prey in experiments with densities up to 40 eggs per arena and no mildew. However, the number of eggs consumed decreased more than twofold when mildew conidia were supplied, even at high prey densities. Differences in predation rate between treatments with and without mildew were highly significant. The results thus indicate that availability of mildew as an alternative food can reduce prey suppression by T. pyri . Possible implications of these findings in biological control of spider mites by generalist predatory mites are discussed.     

Both stable and unstable QTLs for resistance to powdery mildew are detected in apple after four years of field assessments

Powdery mildew, caused by the ascomycete fungus Podosphaera leucotricha, is one of the most damaging diseases of apple worldwide. Polygenically determined resistance might contribute to a significant increase of resistance to this disease in new cultivars. A quantitative trait locus (QTL) analysis was performed in an F₁ progeny derived from a cross between the apple cultivar Discovery and the apple hybrid TN10-8. Powdery mildew incidence was assessed during four years (five seasons) in spring and/or autumn in a French local orchard. Seven additive and/or dominant QTLs were detected over the five seasons, with effects (R ²) ranging from 7.5% to 27.4% of the progeny phenotypic variation. Two QTLs, on linkage groups (LGs) 2 and 13, were consistently identified and accounted together from 29% to 37% of the phenotypic variation according to the year of assessment. The other QTLs were identified during one (LGs 1, 14), two (LG10), or three (LGs 8, 17) seasons. Their instability indicated a changing genetic determinism according to the year of assessment, for which several hypotheses may be put forward. The QTLs on LGs 2 and 8 mapped close to clusters of resistance gene analogs (RGAs) and major genes for resistance to mildew or apple scab previously identified. The stable QTLs identified on LGs 2 and 13, together with the strong effect QTL located on LG 8, are of special interest for breeding purposes, especially if combined with other major resistance genes.     

Effects of varying pollen load on fruit set,seed set and seedling performance in apple and pear

Two apple crosses and one pear cross were carried out using three different pollen densities. In one of the apple crosses, both fruit and seed set decreased when a mixture of pollen and powder (diluted pollen treatment) was used for pollination. Also, seedlings from the undiluted treatment were more vigorous than those originating from pollination with diluted pollen. In the other apple cross, however, no effect on fruit set, seed set or growth in the first year was found. Mean diameter after the second year differed among treatments, but did not seem to be related to pollen density. For pear, the number of good seeds decreased and the number of deaf, probably unfertilized, seeds increased with decreasing pollen density. However, there was no apparent significant effect of pollen density on seedling growth. With respect to scab resistance and mildew attack in both apple crosses, no differences among treatments were found. The results indicate that gametophytic competition may occur in apple crosses, but that it is probably weaker in pear.     

CONCENTRATE SPRAYING OF APPLE TREES I. FUNGICIDAL EFFICIENCY OF LIME-SULPHUR

Experiments are described in which the efficiency of undiluted lime-sulphur applied as a spray mist of small droplets, formed by and carried in a stream of compressed air, was compared on small Cox's Orange Pippin apple trees with that of diluted lime-sulphur applied either in the same way or by orthodox hydraulic spraying. The control without spray damage of apple scab ( Venturia inaequalis (Cooke) Wint.) and apple mildew ( Podosphaera leucotricha (Ell. & Everh.) Salm.) was the criterion.
The undiluted concentrate proved at least as effective and safe as the hydraulically applied dilute spray when a satisfactory method of application, by a paint spray gun, had been established. A 50% dilution applied in this way at increased volume proved equally effective, but a 10% dilution similarly applied failed to give satis-factory control of scab, probably because of the reduced dosage.     

New records of microcyclic conidiogenesis in some powdery mildew fungi

Microcyclic conidiogenesis (MC) was recently described in several species of powdery mildew fungi. This process, defined as the production of conidia on a fungal spore without any, or only a minimal, involvement of hyphal growth, was observed on powdery mildew conidia that have already germinated on host plant surfaces and have been attached to the epidermal cells. Most probably, MC contributes to a quick propagation of young powdery mildew colonies because new conidia are sometimes produced in a shorter time on microcyclic conidiophores than on the hyphae of the young mycelium. This article reports MC in Erysiphe necator ex grapevine, Podosphaera leucotricha ex apple, Golovinomyces orontii ex tobacco, and Neoerysiphe galeopsidis ex Lamium purpureum based on light and low-temperature scanning electron microscopic studies.     

Utilization of pedigree information to estimate genetic parameters from large unbalanced data sets in apple

 Genetic parameters (narrow-sense heritabilities and genetic correlations) were estimated for major agronomical traits in apple from large unbalanced data sets, with the help of wide-pedigree information. The software REML VCE (Groeneveld 1996) took into account the complex pedigree of the French apple-breeding population, thanks to the restricted maximum likelihood procedure combined with the construction of the entire relationship matrix between hybrids planted in the field and their ancestors. Narrow-sense heritability estimates ranged from 0.34 to 0.68 for traits exhibiting a normal distribution. Heritability values around 0.35–0.40 were obtained for fruit characteristics (size, texture, flavour, juice content, attractiveness, russeting). Higher values of heritability were obtained for vigour, assessed by the circumference of the trunk (0.51), and powdery mildew resistance (0.68). Additive genetic correlations between traits were also estimated, showing a very high relationship between fruit-quality traits. Vigour and powdery mildew resistance were slightly correlated with the other traits. Utilization of the ‘individual genetic model’ for the estimation of genetic parameters and breeding values in apple is discussed. Received: 14 June 1997 / Accepted: 4 December 1997     

Effects of some surfactant fungicides on Rhizobium trtfolii and its symbiotic relationship with white clover

Effects of the surfactant apple mildew eradicants Manoxol OT, Alk 3, Triton X45, PP 222, Ethylan CP, Hyamine 3500, Aromox DMMCDW and Aromox C12W on growth and respiration of R. trifolii in vitro and on its symbiotic nitrogen fixation in association with white clover were measured. Most treatments reduced growth up to 8 wk; after 12 wk plant weight and nitrogenase activity were significantly reduced (P = 0.01) by Triton X45, Manoxol OT and PP 222 at concentrations of 1000, 2500 and 5000 μg/gram of soil respectively. Electron microscopy showed that bacteroids in root nodules where nitrogen fixation was inhibited had contracted away from the host membrane leaving an intracellular space. Damage to the bacteroid membrane was apparent. Surfactants had no demonstrable effect on adhesion of the bacteria to root hairs. In apple orchards run-off of surfactant fungicides, applied to apple trees at the recommended rates is unlikely to have adverse effects on Rhizobium nodulated white clover.     

Fungitoxic and phytotoxic effect of some surface-active agents applied for the control of apple powdery mildew

Mixtures of methyl esters of fatty acids (‘Off-Shoot O’) and mixtures of fatty alcohols (‘Off-Shoot T’), applied during the late autumn (November), eradicated apple powdery mildew (caused by Podosphaera leucotricha) from infected buds. Applications in the spring at bud-burst were less effective. The autumn sprays caused little damage to the cultivars Bramley's Seedling, Cox's Orange Pippin and Golden Delicious but on Worcester Pearmain 50% of the buds were killed by a 5% Off-Shoot O spray. The bud-burst sprays were damaging and reduced yield on the Cox and Bramley trees. A 5% a.i. mixture of nonanol/lissapol applied to a range of commercial cultivars was effective in eradicating powdery mildew but was more phytotoxic than the Off-Shoot compounds. Spraying Cox trees later in the winter failed to reduce the damage. Eradication of the overwintering stage of the disease resulted in low spore concentrations during the following spring and early summer, enabling some economies to be made in the summer mildew fungicide programme.     

Host Phenology and Geography as Drivers of Differentiation in Generalist Fungal Mycoparasites

The question as to why parasites remain generalist or become specialist is a key unresolved question in evolutionary biology. Ampelomyces spp., intracellular mycoparasites of powdery mildew fungi, which are themselves plant pathogens, are a useful model for studies of this issue. Ampelomyces is used for the biological control of mildew. Differences in mycohost phenology promote temporal isolation between sympatric Ampelomyces mycoparasites. Apple powdery mildew (APM) causes spring epidemics, whereas other powdery mildew species on plants other than apple cause epidemics later in the season. This has resulted in genetic differentiation between APM and non-APM strains. It is unclear whether there is genetic differentiation between non-APM Ampelomyces lineages due to their specialization on different mycohosts. We used microsatellites to address this question and found no significant differentiation between non-APM Ampelomyces strains from different mycohosts or host plants, but strong differentiation between APM and non-APM strains. A geographical structure was revealed in both groups, with differences between European countries, demonstrating restricted dispersal at the continent scale and a high resolution for our markers. We found footprints of recombination in both groups, possibly more frequent in the APM cluster. Overall, Ampelomyces thus appears to be one of the rare genuine generalist pathogenic fungi able to parasitize multiple hosts in natural populations. It is therefore an excellent model for studying the evolution of pathogens towards a generalist rather than host-specific strategy, particularly in light of the tritrophic interaction between Ampelomyces mycoparasites, their powdery mildew fungal hosts and the mildew host plants.     

The role of the apple spray programme in the protection of fruit buds from powdery mildew

Comparisons of programmes, which differed in the dates on which high-volume sprays of dinocap (0.019 %) were omitted, indicated that applications in the period from green cluster to early fruitlet protected the bourse buds of apple cv. Cox's Orange Pippin from infection, and so controlled the number of mildewed blossom trusses in the following year. This role of the sprays in preventing primary mildew declined after the early fruitlet stage. The existence of two phases of the disease was confirmed, and although dinocap at late blossom and early fruitlet contributed to the control of preliminary infections of secondary mildew on vegetative shoots, the latter phase did not become severe until after the peak invasion of bourse buds. Fewer than 20% of the fruit buds which produced primary-mildewed blossom in 1968 were succeeded by healthy fruit buds in the next year; this poor recovery was not improved by dinocap applied during the flowering period in 1968.
Fruit-set and crop of James Grieve and Cox's Orange Pippin were reduced in 1968 by dinocap applied at green cluster and pink bud, or at full blossom and petal-fall, but no deleterious effects were produced by equivalent sprays in 1969. The need for mildew control during flowering is discussed in relation to phytotoxic sensitivity.     

Application of a high-speed breeding technology to apple (Malus × domestica) based on transgenic early flowering plants and marker-assisted selection

Breeding of apple (Malus × domestica) remains a slow process because of protracted generation cycles. Shortening the juvenile phase to achieve the introgression of traits from wild species into prebreeding material within a reasonable time frame is a great challenge. In this study, we evaluated early flowering transgenic apple lines overexpressing the BpMADS4 gene of silver birch with regard to tree morphology in glasshouse conditions. Based on the results obtained, line T1190 was selected for further analysis and application to fast breeding. The DNA sequences flanking the T-DNA were isolated and the T-DNA integration site was mapped on linkage group 4. The inheritance and correctness of the T-DNA integration were confirmed after meiosis. A crossbred breeding programme was initiated by crossing T1190 with the fire blight-resistant wild species Malus fusca. Transgenic early flowering F(1) seedlings were selected and backcrossed with 'Regia' and 98/6-10 in order to introgress the apple scab Rvi2, Rvi4 and powdery mildew Pl-1, Pl-2 resistance genes and the fire blight resistance quantitative trait locus FB-F7 present in 'Regia'. Three transgenic BC'1 seedlings pyramiding Rvi2, Rvi4 and FB-F7, as well as three other BC'1 seedlings combining Pl-1 and Pl-2, were identified. Thus, the first transgenic early flowering-based apple breeding programme combined with marker-assisted selection was established.     

The curative activity of non-ionic surface-active agents against some powdery mildew diseases

Eight non-ionic surfactants of the general formula RO(C₂H₄O)_nH were examined as fungicides for the control of various powdery mildew diseases. Those based on the condensation products of cetyl and oleyl alcohol with ethylene oxide were effective only as curative fungicides, having very little protective potential. This activity was greatest in products with n= 6–10. The most effective derivative, having eight ethylene oxide units, was particularly active in greenhouse tests against Sphaerotheca fuliginea on cucumber, S. pannosa on roses, and Podosphaera leucotricha on apples at concentrations which were not damaging to these crops. Although this compound caused damage to apples in the field it was effective against rose mildew and apple mildew, and its purely curative action gave a level of control equal to the standard dinitro compounds, which are capable of acting as either protectant or curative fungicides. This suggests that the dinitro-compounds also probably act mainly in a curative fashion against these diseases in the field.