QTL mapping of fire blight resistance in apple

Fire blight caused by the bacterium Erwinia amylovora is a severe threat to apple and pear orchards worldwide. Apple varieties exhibit a wide range of relative susceptibility/tolerance to fire blight. Although, no monogenic resistance against fire blight has been identified yet, recent evidence indicates the existence of quantitative resistance. Potential sources of fire blight resistance include several wild Malus species and some apple cultivars. F1 progenies of ‘Fiesta’בDiscovery’ were inoculated with the Swiss strain Ea 610 and studied under controlled conditions to identify quantitative trait loci (QTLs) for fire blight resistance. Disease was evaluated at four time points after inoculation. Shoot lesion length and the area under disease progress curve (AUDPC) values were used for QTL analysis. One significant (LOD score of 7.5–8.1, p<0.001) QTL was identified on the linkage group 7 of ‘Fiesta’ (F7). The F7 QTL explained about 37.5–38.6% of the phenotypic variation.     

Ice formation and tissue response in apple twigs

Abstract. The response of apple twig tissue to a freezing stress was examined using a combination of low temperature scanning electron microscopy and freeze substitution techniques. Bark and wood tissues responded differently. In the bark, large extracellular ice crystals were observed in the cortex. The adjacent cortical cells collapsed and a large reduction in cell volume was observed. The extent of cell collapse throughout the bark was not uniform. Cells in the periderm, phloem and cambium exhibited little change in cell volume compared to cortical cells. Large extracellular ice crystals were not observed in the xylem or pith tissues. The xylem ray parenchyma and pith cells did not collapse in response to a freezing stress, but retained their original shape. The pattern of ice formation and cell response was not observed to change with season or the level of cold acclimation. This study supported the concept that bark and xylem tissues exhibit contrasting freezing behaviour. The observations were consistent with the idea that water in bark freezes extracellularly while water in xylem ray parenchyma and pith cells may supercool to temperatures approaching –40 °C prior to freezing intracellularly.     

Phytoalexin formation in cell suspensions of Phaseolus vulgaris in response to an extract of bean hypocotyls

Suspension cultures of Phaseolus vulgaris accumulated phytoalexins after treatment with an extract of bean hypocotyls. Maximum production of phaseollin occurred during the early exponential phase of culture growth. Phaseollin was converted to phaseollinosoflavan by these cultures and this conversion occurred during accumulation of the phytoalexins. Factors affecting phytoalexin accumulation in these cultures are discussed.     

Phytoalexin induction in plants of tropical environment

Phytoalexin induction, so far reported mainly for cultivated plants, is applied to wild species of tropical environment. The phenomenon is strongly influenced by the experimental techniques. Application of the more common drop diffusate procedure results in the predominance of positive phytoalexin responses in cultivated (rather than in wild) species, while the facilitated diffusion procedure leads to comparable results for cultivated and wild species. Positive phytoalexin responses by both procedures are more common in the rainy (spring and summer) than in the dry (autumn and winter) season.     

Nutrient status of apple blossoms and their susceptibility to fire blight

Blossoms of Malus sylvestris cv. Golden Delicious trees on M. 7 root-stocks, maintained under various N-P-K nutrient regimes in sand culture in the glasshouse, were inoculated with Erwinia amyhvora, the incitant of fire blight. Widest differences between nutrient treatments occurred when about ten E. amylovora cells were applied to each blossom. Trees with high N and high P contents had most infections. Trees with lower P content were less susceptible. Of trees with low P content, those with higher K appeared more susceptible.     

Timing of the phases in adventitous root formation in apple microcuttings

As with other regeneration processes, adventitious root formationmay be divided into three phases, namely dedifferentiation,induction and differentiation. We assumed that the appropriatehormonal conditions for rooting, in particular a high levelof auxin and a low level of cytokinin, are required only duringthe induction phase. Hence, the effect of 24 h pulses with indolebutyricacid (IBA) or benzylaminopurine (BAP) should be maximal duringthis phase. On this assumption, the timing of the three phaseswas determined in microcuttings of Malus ‘Jork 9’.The promotion of rooting was largest for 24 h IBA-pulses given24–48 h or 48–72 h after the onset of culture. Duringculture of shoots on IBA-containing medium 24 h BAP-pulses weregiven. Inhibition of rooting was maximal for the BAP-pulsesgiven between 24 and 96 h. An analysis of the kinetics of rootemergence also indicated that added IBA acted from 24 h onwards:emergence of roots from the tissue occurred simultaneously forthe IBA pulses at 0–24 h and 24–48 h, but lagged1 d behind for the 48–72 h pulse and 2 d for the 72–96h pulse. We concluded that dedifferentiation occurred from 0to 24 h, induction from 24 to 72 or 96 h, and after that differentiation.Histological observations showed that after 24 h, cells withswollen nuclei and dense cytoplasm had appeared in the regionsof the stem where the roots were formed. The first cell divisionswere observed after 48 h. After 96 h, meristemoids of c. 30cells had been formed. After the BAP-pulses at 24–48 hor 48–72 h, these meristematic cells formed callus. Key words: Adventitious rooting, apple, auxin, cytokinin, Malus, regeneration.     

Phytoalexin production in lucerne roots inoculated withVerticillium albo-atrum

Summary Out of various soil and plant test methods tested for predicting response of rice to K application in soils of a rice growing valley region the Hanway and Heidal extractant neutralN NH₄ OAc turned out to be the best. The critical limit of extractable K was 160 ppm by the Hanway and Heidal extractant, and by the Bray's reagent 175 ppm. Critical K level in the rice plant is 0.4%. Correlations between the extractable K and K uptake were highly positive for various extractants: Hanway and Heidal, Morgan, Hunter and Pratt No. 2, Blanchet and Perigand and MacLean. Although majority of the soils of the region was Inceptisols followed by Alfisols and Vertisols, all soil types had a similar available nutrient status and a similar pattern in relative grain yields. K response was noticeable in Alfisols with respect to grain and straw yields. The grain P concentration in Vertisols, and straw K in Alfisols indicated the contribution of K towards the productivity of two soil groups.     

The fire blight pathogen Erwinia amylovora requires the rpoN gene for pathogenicity in apple

RpoN is a σ⁵⁴ factor regulating essential virulence gene expression in several plant pathogenic bacteria, including Pseudomonas syringae and Pectobacterium carotovorum. In this study, we found that mutation of rpoN in the fire blight pathogen Erwinia amylovora caused a nonpathogenic phenotype. The E. amylovora rpoN Tn5 transposon mutant rpoN1250::Tn5 did not cause fire blight disease symptoms on shoots of mature apple trees. In detached immature apple fruits, the rpoN1250::Tn5 mutant failed to cause fire blight disease symptoms and grew to population levels 12 orders of magnitude lower than the wild‐type. In addition, the rpoN1250::Tn5 mutant failed to elicit a hypersensitive response when infiltrated into nonhost tobacco plant leaves, and rpoN1250::Tn5 cells failed to express HrpN protein when grown in hrp (hypersensitive response and pathogenicity)‐inducing liquid medium. A plasmid‐borne copy of the wild‐type rpoN gene complemented all the rpoN1250::Tn5 mutant phenotypes tested. The rpoN1250::Tn5 mutant was prototrophic on minimal solid and liquid media, indicating that the rpoN1250::Tn5 nonpathogenic phenotype was not caused by a defect in basic metabolism or growth. This study provides clear genetic evidence that rpoN is an essential virulence gene of E. amylovora, suggesting that rpoN has the same function in E. amylovora as in P. syringae and Pe. carotovorum.     

Factors Affecting Phytoalexin Production in Cultured Carrot Cells

The production of 6-methoxymellein, a phytoalexin, in culturedcarrot cells under various growth conditions was studied usingtwo induction methods; by adding partial hydrolysates obtainedby treating the cells with pectinase or trypsin, or by directlyadding these enzymes to growing cells to cause the release ofcellular components as endogenous elicitor. 6-Methoxymellein production depended greatly on the cell cultureage. Maximal production was found in cells at the early stationaryphase, while actively dividing cells had only negligible amounts.Release of elicitor from the cells by pectinase or trypsin wasalso influenced by the culture stage. Effective elicitor wasobtained only from cells in the late logarithmic and early stationaryphases. 6-Methoxymellein production required the presence of 2,4-D.IAA could not substitute for 2,4-D, though partial hydrolysatesprepared from these cells grown with IAA or without auxin showedsignificant elicitor activity. On the other hand, the productionwas inhibited by actinomycin D or cycloheximide, suggestingthat de novo syntheses of RNA and protein are required for thephytoalexin production. (Received November 17, 1984; Accepted March 2, 1985)     

Phytoalexin production by Ononis species

Following exposure to short wavelength (254 nm) ultra-violet light, the detached leaflets of Passaea (Ononis) ornithopodioides and 31 species and subspecies of Ononis have been found to accumulate substantial quantities of the isoflavonoid (pterocarpan) phytoalexin, medicarpin. Apart from P. (Ononis) ornithopodioides, O. cristata, O. fruticosa, O. pubescens and O. rotundifolia, leaf tissues of all the species investigated similarly contained small amounts of the related fungitoxic pterocarpan, maackiain. Isoflavan phytoalexins common in genera such as Medicago and Trifolium (tribe Trifolieae) were absent from both Ononis and Passaea. The phytoalexin data suggest that Passaea should probably be combined with Ononis and, in conjunction with information on constitutive isoflavonoids, that Ononis itself should be assigned to the Trifolieae rather than to the distinct tribe Ononideae. Chemical evidence for and against an especially close taxonomic association between Ononis and Cicer (tribe Cicereae) is also briefly discussed.     

Influence of light on ascorbate formation and metabolism in apple fruits

To further understand the regulatory mechanism of light on the formation of ascorbic acid (AsA) in the sink organs of plants, a systematical investigation on AsA levels, activities of two key biosynthsis enzymes and their mRNA expression as well as the recycling was performed in the fruits of apple (Malus domestica Borkh), under different levels of shade. After the whole trees were shaded with the sun-light about 50–55% for 20 days, AsA levels were significantly decreased in fruit peel, flesh and leaves, while mRNA expression levels and activities of l-galactose dehydrogenase (l-GalDH, EC 1.1.1.117) and l-galactono-1,4-lactone dehydrogenase (l-GalLDH, EC 1.3.2.3) as well as activities of recycling enzymes was clearly declined in the leaf and peel but not in the flesh. By shading fruits only for 20 days, AsA levels, relative mRNA levels and activities of l-GalDH and l-GalLDH as well as activities of recycling enzymes all showed obvious decrease in the peel, but not in the flesh. However, their levels in the peel were markedly increased after the full shade was removed and re-exposed these fruits on natural light for 5 days. It is concluded that light affects AsA biosynthesis and recycling in the peel and leaf, but did not in the fresh. Results also suggest that apple fruit is potential to biosynthesize AsA via the l-galactose pathway, and AsA content in the fruits may depend partly on levels of AsA or other photochemistry controlled by light in the leaves.     

Ethylene inhibitors enhance in vitro root formation from apple shoot cultures

Effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and three ethylene inhibitors, AgNO₃, aminoethoxyvinyglycine (AVG) and CoCl₂, on root formation were tested in vitro using shoot cultures of the apple (Malus×domestica Borkh.) cultivar Royal Gala. ACC inhibited root formation by delaying root emergence and increasing callus formation at the bases of shoots. In contrast, ethylene inhibitors promoted root formation. Both AgNO₃ and AVG at the appropriate concentrations increased the percentage of shoots producing roots and reduced callus formation at the base of these shoots. AgNO₃ stimulated root emergence and enhanced root growth, while AVG increased the number of roots per shoot. CoCl₂ slightly increased root number and rooting efficiency. These promotive effects may result from a reduction in ethylene concentration or inhibition of ethylene action. The results found in this study may be used to improve the rooting efficiency of other apple cultivars and rootstocks, and possibly of other plant species. Received: 2 March 1997 / Revision received: 1 July 1997 / Accepted: 18 July 1997     

Nitric Oxide Induces Phytoalexin Accumulation in Potato Tuber Tissues

We investigated whether nitric oxide (NO) radical could inducephytoalexin production. Treatment of potato tuber tissues with1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC-18), anNO-releasing compound, induced the accumulation of the potatophytoalexin rishitin. This induction was inhibited by carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(carboxy-PTIO), an NO-specific scavenger, or by Tiron, a radicalscavenger, suggesting a phytoalexin inducing activity for NO. (Received December 7, 1995; Accepted January 4, 1996)     

Protection of apple against fire blight induced by an hrpL mutant of Erwinia amylovora

A regulatory hrpL non-virulent mutant of Erwinia amylovora is effective in controlling fire blight disease when inoculated on apple seedlings simultaneously with the pathogenic parental strain. Mechanisms involved in this protective effect were investigated. The use of two marker genes, uidA and lacZ, expressed in the hrpL mutant and the pathogenic strain, respectively, allowed to localize simultaneously the two inoculated strains in plant tissue. An anti-β-glucuronidase antibody was also used to detect the hrpL mutant. Both techniques indicated that the two strains localized mainly in separate areas of the leaf tissue. In addition, leaves infiltrated with the hrpL mutant exhibited a significant increase in peroxidase activity in contrast to a hrp secretion mutant known to be less effective in the protection. It is suggested that protection obtained with the hrpL mutant relies on the physical separation between the mutant and the parental strain after co-inoculation and the rapid and sustained activation of plant defense mechanisms in reactive tissue, i.e. not invaded by the virulent strain.     

Induction of Phytoalexin Formation in Suspension-cultured Rice Cells by N-Acetyl-chitooligosaccharides

Induction of phytoalexin formation in suspension-cultured rice cells by a series of N-acetylchitooligosaccharides and chitooligosaccharides was studied. N-acetylchitooligosaccharides larger than hexaose induced the formation of momilactones A and B as well as oryzalexins A, B, and D at very low concentrations like 10^{? 9}–10^{? 6} M (N-acetylchitoheptaose). GlcNAc oligomers smaller than trimers had almost no activity and a series of deacetylated chitooligosaccharides were also inactive. Strict requirement for the size and structure of GlcNAc oligomers as well as the sensitivity to them strongly indicates the presence of recognition systems specific for these compounds in rice cells. The level of momilactone A produced reached 100–500 μg/g of cultured cells, which appeared to be enough to prevent the growth of pathogenic fungi such as Pyricularia oryzae, thus indicating the importance of this phenomenon in the defense systems of rice plants. Suspension-cultured cells obtained only from a suitable period of cultivation, mainly those from lag phase, could respond to the elicitor and produce phytoalexins.     

Transgenic apple plants overexpressing the Lc gene of maize show an altered growth habit and increased resistance to apple scab and fire blight

Transgenic apple plants (Malus × domestica cv. ‘Holsteiner Cox’) overexpressing the Leaf Colour (Lc) gene from maize (Zea mays) exhibit strongly increased production of anthocyanins and flavan-3-ols (catechins, proanthocyanidins). Greenhouse plants investigated in this study exhibit altered phenotypes with regard to growth habit and resistance traits. Lc-transgenic plants show reduced size, transversal gravitropism of lateral shoots, reduced trichome development, and frequently reduced shoot diameter and abnormal leaf development with fused leaves. Such phenotypes seem to be in accordance with a direct or an indirect effect on polar-auxin-transport in the transgenic plants. Furthermore, leaves often develop necrotic lesions resembling hypersensitive response lesions. In tests, higher resistance against fire blight (caused by the bacterium Erwinia amylovora) and against scab (caused by the fungus Venturia inaequalis) is observed. These phenotypes are discussed with respect to the underlying altered physiology of the Lc-transgenic plants. The results are expected to be considered in apple breeding strategies.     

Multicellular spheroid formation and evolutionary conserved behaviors of apple snail hemocytes in culture

A hemocyte primary culture system for Pomacea canaliculata in a medium mimicking hemolymphatic plasma composition was developed. Hemocytes adhered and spread onto culture dish in the first few hours after seeding but later began forming aggregates. Time-lapse video microscopy showed the dynamics of the early aggregation, with cells both entering and leaving the aggregates. During this period phagocytosis occurs and was quantified. Later (>4 h), hemocytes formed large spheroidal aggregates that increased in size and also merged with adjacent spheroids (24–96 h). Large single spheroids and spheroid aggregates detach from the bottom surface and float freely in the medium. Correlative confocal, transmission electron and phase contrast microscopy showed a peculiar organization of the spheroids, with a compact core, an intermediate zone with large extracellular lacunae and an outer zone of flattened cells; also, numerous round cells emitting cytoplasmic extensions were seen attaching to the spheroids' smooth surface. Dual DAPI/propidium iodide staining revealed the coexistence of viable and non-viable cells within aggregates, in varying proportions. DNA concentration increased during the first 24 h of culture and stabilized afterward. BrdU incorporation also indicated proliferation. Spontaneous spheroid formation in culture bears interesting parallels with spheroidal hemocyte aggregates found in vivo in P. canaliculata, and also with spheroids formed by tumoral or non-tumoral mammalian cells in vitro.