Susceptibility of selected apple cultivars to the Mediterranean fruit fly

Ceratitis capitata (Wiedemann), the Mediterranean fruit fly, is one of the key pest species affecting deciduous fruit orchards along the Mediterranean coasts. Because of global warming, C. capitata is gradually spreading north and is becoming a major pest of apples. Determining the susceptibility of the main apple varieties grown in the region will serve as a cornerstone to the management of this pest. In this study, we show the results of a field and laboratory no‐choice test conducted to determine the Medfly preferences on different apple cultivars. The seven main varieties of apples (Gala, Red Delicious, Golden Delicious, Granny Smith, Kanzi, Morgen Dallago and Fuji) were tested. The results demonstrate that C. capitata lays eggs on all apple cultivars in both field and laboratory conditions. The Granny Smith, Red Delicious and Morgen Dallago varieties showed the lowest susceptibility in laboratory conditions, (0.75, 1.55, 2 oviposition punctures/fruit, respectively), with significant differences in oviposition compared to the Golden Delicious, Kanzi and Fuji (3.27, 3.31, 3.1 oviposition punctures/fruit, respectively) varieties, which were shown to be the most susceptible to Medfly attack in laboratory conditions. On the other hand, only slight and not statistically significant differences emerged from the field trials. In relation to the physico‐chemical characteristics, the apple cultivars showing the lowest susceptibility (Granny Smith, Red Delicious and Morgen Dallago) had harder peels and pulps and lower sugar contents than the most susceptible cultivars (Golden Delicious, Fuji and Kanzi). These results were also confirmed through evaluation of larval development on different varieties. In fact, Granny Smith, Red Delicious and Morgen Dallago were the three varieties that did not allow adequate larval and adult development and reduced the possibility of the emergence of a new generation.     

Significance of skin flavonoids for UV-B-protection in apple fruits

An attempt has been made to assess the UV-B-protective capacity of phenolic compounds accumulated in superficial structures of plants using apple fruit as a model. Two apple (Malus domestica Borkh.) cultivars (Braeburn and Granny Smith) differing in response to high fluxes of solar radiation were selected and exposed to increasing doses of UV-B radiation. The extent of UV-B-induced damage to photosystem II of apple skin correlated with its quercetin glycoside (but not anthocyanin) content. Granny Smith apples did not demonstrate a pronounced response to high sunlight in terms of the accumulation of phenolic substances in the skin and exhibited similar patterns of Fo, Fm, and Fv/Fm changes in the course of UV-B irradiation both on sun-exposed and shaded surfaces of a fruit. Unlike Granny Smith, Braeburn fruits were characterized by a significant accumulation of quercetin glycosides in sun-exposed skin, however, shaded skin contained these compounds in similar amounts to those in Granny Smith. Accordingly, photosystem II in sun-exposed skin of Braeburn apples was resistant to high doses of UV-B radiation (up to 97 kJ m-2), whereas the susceptibility of the photosynthetic apparatus in shaded skin of Braeburn to UV-B-induced damage was much higher and similar to that of both sun-exposed and shaded skin of Granny Smith fruits. Anthocyanins, at least in the amounts found in Braeburn, did not show an additional effect in UV-B protection.     

Differential gene expression analysis of ‘Granny Smith’ apple (Malus domestica Borkh.) during fruit skin coloration

‘Granny Smith’ apples growing under normal sunlight develop green skin, whereas the peel turns red due to anthocyanin accumulation after the removal of a bagging treatment. Two anthocyanins, Cyanidin 3-O-galactoside (cy3-gal) and Cyanidin 3-O-arabinoside (cy3-ara), were detected in the red ‘Granny Smith’ apple peels, and cy3-gal was determined to be chiefly responsible for the red color. The content of cy3-gal was more than 98% of the total anthocyanin in the red ‘Granny Smith’ peels. To better understand the molecular basis of anthocyanin biosynthesis in ‘Granny Smith’ apples, we performed a quantitative real-time PCR (qRT-PCR) analysis of anthocyanin biosynthetic genes (MdCHS, MdF3H, MdDFR, MdANS, MdUFGT, and MdMYB1). Our results indicate that the expression of these genes (except MdCHS) was associated with increased anthocyanin accumulation in the skin of ‘Granny Smith’ apples. Four selected genes obtained from the ‘Granny Smith’ skin cDNA library, phytoene synthase (PSY), WD40 repeat protein, polygalacturonase (PG), and galactosidase (GAL), were also confirmed by qRT-PCR. We found that these genes were differently expressed during ‘Granny Smith’ apple skin coloration, suggesting that they are directly or indirectly involved in pigment accumulation. In conclusion, anthocyanin biosynthesis in ‘Granny Smith’ apples is the result of interactions between multiple enzymes in the anthocyanin biosynthesis pathway, and the coloring mechanism of ‘Granny Smith’ apples may be similar to that of red-skinned cultivars.     

A test of fruit varieties on entry rate and development by neonate larvae of the codling moth,Cydia pomonella

The rate of entry by neonate larvae of the frugivorous codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), into fruit material was investigated. We used no‐choice bioassays in climate‐controlled rooms to assay larval entry across four host plant species (apple, pear, quince, walnut) and three varieties within a single fruit species (apple). Larvae successfully entering apples were reared to adulthood, and we collected tissue samples from apples which were successfully colonized in order to determine sucrose concentrations. This information was used to evaluate differences in adult moth size, development time, and pulp sucrose concentration due to apple variety. Four important findings emerged: (1) neonate larvae had the highest frequency of entry (86% of larvae) into apple fruits, compared with pear (78%), quince (56%), and walnut (32%); (2) the frequency of larval entry into immature apples differed across apple varieties, and larval entry rate was highest in variety Golden Delicious (72%), compared with Granny Smith (46%) and Red Delicious (64%); (3) on average, adult moths were larger and development times were shorter on the variety with the highest entry frequency (Golden Delicious); and (4) apple pulp sucrose concentrations were higher for Golden Delicious (17.5 μg mg^?1) than for either Granny Smith (15.9 μg mg^?1) or Red Delicious (15.1 μg mg^?1) varieties, which correlates positively with entry and development data. We conclude that host fruit species and varietals within a species affect the entry rate of neonate codling moth larvae in no‐choice assays. We hypothesize that larval development is influenced by mean sucrose concentrations or other phytochemical differences associated with host fruit varieties.     

Molecular cloning and characterization of constans-like cDNA clones of the fuji apple

Two cDNA clones,MdCOL1 andMdCOL2, encoding CONSTANS (CO)-like proteins were isolated from an apple (Malus domestica cv. Fuji) fruit cDNA library. Both proteins contain two zinc finger motifs at the amino terminal end and a putative nuclear localization domain at the carboxyl terminal end. Genomic DNA blot analysis suggests that theCO-like genes are members of a small multigene family. RNA blot and RT-PCR analyses revealed that these genes are expressed in every organ that was examined. However, the expression levels were higher in floral buds and fruits at their early developmental stages compared to late reproductive stages or vegetative organs. Such expression patterns are quite different from those of theCO-like genes fromArabidopsis, which show strong organ specificity in either roots, cauline leaves, or flowers. These results indicate that the appleCO-like genes are significantly different from theArabidopsis genes and that they appear to play important roles in reproductive organ development.     

Laboratory tests carried out onMicrothrix inconspicuella [Lep.: Pyralidae], a biological control agent forEmex australis in Australia

Host specificity tests of the moth,Microthrix inconspicuella Ragonot in Australia, indicated that larvae could feed and develop on young apple leaves. Additional tests in South Africa on leaves and fruit of the 4 apple varieties, Jonathan, Starking (Red Delicious), Granny Smith and Golden Delicious, showed that apples were not a preferred food. Little feeding occurred and pupation happened infrequently. No 2^nd generation resulted whenM. inconspicuella colonies were confined on apple fruit or leaves.      

Apple beta-galactosidase. Activity against cell wall polysaccharides and characterization of a related cDNA clone.

A beta-galactosidase was purified from cortical tissue of ripe apples (Malus domestica Borkh. cv Granny Smith) using a procedure involving affinity chromatography on lactosyl-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that two polypeptides of 44 and 32 kD were present in the fraction that showed activity against the synthetic substrate p-nitrophenol-beta-D-galactopyranoside. The enzyme preparation was incubated with polysaccharide extracts from apple cell walls containing beta-(1-->4)-linked galactans, and products of digestion were analyzed by gas chromatography. Small amounts of monomeric galactose were released during incubation, showing that the enzyme was active against native substrates. Amino acid sequence information was obtained from the purified protein, and this showed high homology with the anticipated polypeptide coded by the ethylene-regulated SR12 gene in carnation (K.G. Raghothama, K.A. Lawton, P.B. Goldborough, W.R. Woodson [1991] Plant Mol Biol 17: 61-71) and a harvest-related pTIP31 cDNA from asparagus (G. King, personal communication). Using the asparagus cDNA clone as a probe, an apple homolog (pABG1) was isolated. This clone contains a 2637-bp insert, including an open reading frame that codes for a polypeptide of 731 amino acids. Cleavage of an N-terminal signal sequence would leave a predicted polypeptide of 78.5 kD. Genomic DNA analysis and the isolation of other homologous apple clones suggest that pABG1 represents one member of an apple beta-galactosidase gene family. Northern analysis during fruit development and ripening showed accumulation of pABG1-homologous RNA during fruit ripening. Enzyme activity as measured in crude extracts increased during fruit development to a level that was maintained during ripening.     

桃ACC合酶基因的分离及其差异表达

利用5′/3′RACE PCR技术,从桃(Prunus persica (L.) Batsch)果实中克隆了植物乙烯生物合成的关键酶--ACC合酶的全长cDNA pacs,对pacs基因进行全序列测定表明,该基因全长1 848个碱基,编码区为1 449个碱基,5′端有177个碱基的非编码区序列,3′端有219个碱基的非编码区序列(不包括终止密码子TAA).pacs基因编码区共编码483个氨基酸,蛋白质大小为54 kD,等电点为6.43.pacs与番茄(S19677)、梅(AB031026)、番木瓜(U68216)、苹果(AB034993)等其他植物ACC合酶cDNA氨基酸序列同源性分别为65%、70%、75%、90%,并存在与这些ACC合酶氨基酸的活性位点保守序列SLSKDMGFPGFR.RT-PCR结合杂交分析表明,pacs和我们以前克隆的桃ACC合酶cDNA pacs12(AF467782)在叶片和花中基因表达模式基本一致,伤处理和IAA均能诱导叶片pacs 和pacs12基因的表达,但pacs在伤处理叶片的表达水平比pacs12高;pacs 和pacs12基因在果实表达有所不同,pacs在绿熟和成熟果实中均有表达,而pacs12在绿熟果实中基本检测不到,在成熟果实中才有表达,两者在果实中的表达水平比伤处理和IAA处理叶片和花中要低.     

Patterns of pigment changes in apple fruits during adaptation to high sunlight and sunscald development

Reflectance spectra of four apple (Malus domestica Borkh.) cultivars were studied and chlorophyll, carotenoid, anthocyanin and flavonoid content in sunlit and shaded peel was determined. In all cases sunlit peel accumulated high amounts of phenolics (flavonoid glycosides). Adaptation to strong sunlight of an apple cultivar with limited potential for anthocyanin biosynthesis (Antonovka) was accompanied by a decrease in chlorophyll and a significant increase in total carotenoid content. The increase in carotenoids also took place in sunlit sides of the Zhigulevskoye fruits, accumulating high amounts of anthocyanins, but chlorophyll content in sunlit peel was higher than that in shaded peel. Significant increases in carotenoids and anthocyanins were detected during fruit ripening when chlorophyll content fell below 1.5–1.8 nmol cm^–2. Chlorophyll in sunlit fruit surfaces of both cultivars was considerably more resistant to photobleaching than in shaded (especially of Zhigulevskoye) sides. Induced by sun irradiation, the photoadaptive responses were cultivar-dependent and expressed at different stages of fruit ripening even after storage in darkness. The development of sunscald symptoms in susceptible apple cultivars (Granny Smith and Renet Simirenko) led to a dramatic loss of chlorophylls and carotenoids, which was similar to that observed during artificial photobleaching. The results suggest that apple fruits exhibit a genetically determined strategy of adaptation of their photoprotective pigments to cope with mediated by reactive oxygen species photodynamic activity of chlorophyll under strong solar irradiation. This includes induction of synthesis and accumulation of flavonoids, anthocyanins and carotenoids that could be expressed, if necessary, at different stages of fruit development     

Expression profiles of genes encoded by the supernumerary chromosome controlling AM-toxin biosynthesis and pathogenicity in the apple pathotype of Alternaria alternata

The apple pathotype of Alternaria alternata produces host-specific AM-toxin and causes Alternaria blotch of apple. Previously, we cloned two genes, AMT1 and AMT2, required for AM-toxin biosynthesis and found that these genes are encoded by small, supernumerary chromosomes of <1.8 Mb in the apple pathotype strains. Here, we performed expressed sequence tag analysis of the 1.4-Mb chromosome encoding AMT genes in strain IFO8984. A cDNA library was constructed using RNA from AM-toxin-producing cultures. A total of 40,980 clones were screened with the 1.4-Mb chromosome probe, and 196 clones encoded by the chromosome were isolated. Sequence analyses of these clones identified 80 unigenes, including AMT1 and AMT2, and revealed that the functions of 43 (54%) genes are unknown. The expression levels of the 80 genes in AM-toxin-producing and nonproducing cultures were analyzed by real-time quantitative polymerase chain reaction (PCR). Most of the genes were found to be expressed in both cultures at markedly lower levels than the translation elongation factor 1-alpha gene used as an internal control. Comparison of the expression levels of these genes between two cultures showed that 21 genes, including AMT1 and AMT2, were upregulated (>10-fold) in AM-toxin-producing cultures. Two of the upregulated genes were newly identified to be involved in AM-toxin biosynthesis by the gene disruption experiments and were named AMT3 and AMT4. Thus, the genes upregulated in AM-toxin-producing cultures contain ideal candidates for novel AM-toxin biosynthetic genes.     

Relationship of Endopolygalacturonase Inhibitor Activity to the Rate of Fungal Rot Development in Apple Fruits

An inhibitor extracted form the cell walls of apple fruits suppressed the activity of endopopygalacruronases (endo-PGs) produced in vivo and in vitro by Nectria galligena, Phomopsis mali, Fusarium Lateritium and Glomerella cingulata but not the endo-PGs produced by Penicillium expansum or Phytophtobora syringae. Of four apple cultivars tested Granny Smith tissue contained the highest levels of inhibitor and Cox's Orange Pippin contained the least. Linear rot expansion in the four apple cultivars inoculated with N. galligena was inversely related to inhibitor activity in the fruit tissue, rot development being slowest in Granny Smith fruits and most rapid in Cox's Orange Pippin fruits. Rot expansion in fruits inoculated with P. expansum bore no such relationship to inhibitor activity in the tissue Apple tissue maceration by the endo-PGs from N. galligena, P. mali. F. lateritium and G. cingulata was similarly related to inhibitory activity in the fruit. The properties of the partially purified inhibitor were consistent with it being proteinaceous but the relative slowness with which it was hear inactivated and the presence of a small percentage of carbohydrate might indicate that it was a glycoprotein.     

Inhibitory effect of diazocyclopentadiene on the development of superficial scald in Granny Smith apple

In this work, diazocyclopentadiene (DACP), an ethylene action inhibitor was used to test if ethylene is involved in the development of superficial scald of apple. Apples (Malus domestica Borkh., c.v. Granny Smith) were pre-stored at 0°C for a month before DACP treatment. After treatment, fruit were stored at 0°C for a further 17 weeks before being transferred to room temperature for a week. The incidence of superficial scald, contents of -farnesene and conjugated triene in fruit skin were analysed. Ethylene production, respiration rate, flesh firmness and soluble solids content of fruit were determined. Results indicated that superficial scald is related to chilling injury. DACP delayed ripening, and dramatically inhibited the development of superficial scald in Granny Smith apple by lowering -farnesene and conjugated triene contents. Ethylene might promote -farnesene synthesis presumably by binding to the ethylene receptor(s).     

The O‐methyltransferase gene MdoOMT1 is required for biosynthesis of methylated phenylpropenes in ripe apple fruit

Phenylpropenes, such as eugenol and trans‐anethole, are important aromatic compounds that determine flavour and aroma in many herbs and spices. Some apple varieties produce fruit with a highly desirable spicy/aromatic flavour that has been attributed to the production of estragole, a methylated phenylpropene. To elucidate the molecular basis for estragole production and its contribution to ripe apple flavour and aroma we characterised a segregating population from a Royal Gala (RG, estragole producer) × Granny Smith (GS, non‐producer) apple cross. Two quantitative trait loci (QTLs; accounting for 9.2 and 24.8% of the variation) on linkage group (LG) 1 and LG2 were identified that co‐located with seven candidate genes for phenylpropene O‐methyltransferases (MdoOMT1–7). Of these genes, only expression of MdoOMT1 on LG1 increased strongly with ethylene and could be correlated with increasing estragole production in ripening RG fruit. Transient over‐expression in tobacco showed that MdoOMT1 utilised a range of phenylpropene substrates and catalysed the conversion of chavicol to estragole. Royal Gala carried two alleles (MdoOMT1a, MdoOMT1b) whilst GS appeared to be homozygous for MdoOMT1b. MdoOMT1a showed a higher affinity and catalytic efficiency towards chavicol than MdoOMT1b, which could account for the phenotypic variation at the LG1 QTL. Multiple transgenic RG lines with reduced MdoOMT1 expression produced lower levels of methylated phenylpropenes, including estragole and methyleugenol. Differences in fruit aroma could be perceived in these fruit, compared with controls, by sensory analysis. Together these results indicate that MdoOMT1 is required for the production of methylated phenylpropenes in apple and that phenylpropenes including estragole may contribute to ripe apple fruit aroma.     

Tissue-specific expression of SORBITOL DEHYDROGENASE in apple fruit during early development

Sorbitol, the primary photosynthate and translocated carbohydrate in apple (Malusxdomestica Borkh.), is converted to fructose by sorbitol dehydrogenase (SDH; EC 1.1.1.14) which is active in apple fruit throughout development. In the apple genome, nine SDH genes have been isolated and their sequences characterized, but their individual expression patterns during apple fruit set and development have not been determined. The objective of this work was to ascertain if SDH genes are differentially expressed and how their patterns of expression may relate to SDH activity in apple seed and cortex during early fruit development. Seed SDH activity was found to be much higher than cortex SDH activity per mg and g fresh weight (FW), and seed SDH activity contributed significantly to whole fruit SDH activity during weeks 2-5 after bloom. Five of the nine SDH genes present in the apple genome were expressed in apple fruit. Two SDH genes, SDH1 and SDH3, were expressed in both seed and cortex tissues. SDH2 expression was limited to cortex, while SDH6 and SDH9 were expressed in seed tissues only. SDH isomeric proteins of different pI values were detected in apple fruit. SDH isomers with pI values of 4.2, 4.8, 5.5, and 6.3 were found in seeds, and SDH isomers with pI values of 5.5, 6.3, 7.3, and 8.3 were found in cortex. The present work is the first to show that SDH is highly active in apple seed and that SDH genes are differentially expressed in seed and cortex during early development.     

Apple polygalacturonase inhibiting protein1 expressed in transgenic tobacco inhibits polygalacturonases from fungal pathogens of apple and the anthracnose pathogen of lupins

Extracts from apple fruit (cultivar "Granny Smith") inhibited the cell-wall degrading polygalacturonase (PG) activity of Colletotrichum lupini, the causal agent of anthracnose on lupins, as well as Aspergillus niger PG. Southern blot analysis indicated that this cultivar of apple has a small gene family of polygalacturonase inhibiting proteins (pgips), and therefore heterologous expression in transgenic tobacco was used to identify the specific gene product responsible for the inhibitory activity. A previously isolated pgip gene, termed Mdpgip1, was introduced into tobacco (Nicotiana tabacum) by Agrobacterium-mediated transformation. The mature MdPGIP1 protein was purified to apparent homogeneity from tobacco leaves by high salt extraction, clarification by DEAE-Sepharose and cation exchange HPLC. Purified MdPGIP1 inhibited PGs from C. lupini and PGs from two economically important pathogens of apple trees, Botryosphaeria obtusa and Diaporthe ambigua. It did not inhibit the A. niger PG, which was in contrast to the apple fruit extract used in this study. We conclude that there are at least two active PGIPs expressed in apple, which differ in their charge properties and ability to inhibit A. niger PG.