Genetic analysis of Cydia pomonella (Lepidoptera: Tortricidae) populations with different levels of sensitivity towards the Cydia pomonella granulovirus (CpGV)

Microsatellite (simple sequence repeats, SSR) and mitochondrial DNA markers were used to assess the structure of European codling moth populations showing different levels of susceptibility towards one of the most important biocontrol agents used in apple production, the Cydia pomonella granulovirus CpGV-M. In 638 C. pomonella individuals from 33 different populations a total of 92 different alleles were scored using six SSR loci. The global estimate of genetic differentiation for all 33 populations was not significantly different from zero, thus indicating a lack of genetic differentiation. AMOVA analysis revealed a very weak but significant variance among C. pomonella populations from different geographic regions, however, no significant variation was evident between CpGV-M resistant or susceptible C. pomonella populations. Sequence analysis of a fragment of the cytochrome oxidase subunit 1 in eight C. pomonella populations resulted in 27 haplotypes, which were grouped in two distinct clusters. Again, no genetic differentiation between CpGV-M resistant and susceptible codling moth populations was detectable. In addition, Structure analysis using microsatellites and association tests with mtDNA haplotypes found neither population-level nor individual correlations associated with CpGV-M resistance. Accordingly, this lack of population structure does not allow discriminating between one or several, separate origins of CpGV-M resistance.     

A study of the effects of ‘Candidatus Phytoplasma mali’ on the psyllid Cacopsylla melanoneura (Hemiptera: Psyllidae)

Cacopsylla melanoneura is a univoltine psyllid vector of ‘Candidatus Phytoplasma mali’, the etiological agent of apple proliferation (AP), a severe disease in European apple orchards. The influence of ‘Ca. P. mali’ on the fitness of C. melanoneura was studied. In the spring of 2007, male-female pairs of field-collected adults were exposed to ‘Ca. P. mali’-infected or healthy ‘Golden Delicious’ apple shoots. Exposure to these diseased shoots did not affect the life span of the adult psyllids. However, significantly fewer eggs were laid on the diseased shoots. Furthermore, fewer of the eggs that were laid on the infected plants hatched. Data suggest a detrimental effect of AP phytoplasma on the fitness of C. melanoneura.     

Patterns in Size-Related Variations in Current-Year Shoot Structure in Eight Deciduous Tree Species

SL ) and leaf display index (LDI: the ratio of leaf area to stem length) of current-year shoots are compared for eight deciduous tree species. Two patterns were found in the relationships. In seven species (Acer mono, Aesculus turbinata, Betula grossa, Carpinus tschonoskii, Fagus crenata, Pterocarya rhoifolia, and Quercus mongolica var. grosseserrata), LDI decreased exponentially with SL, which suggests that short current-year shoots favor leaf-display over space-acquisition, and the reverse for long current-year shoots. The decrease in LDI was much greater and sharper in B. grossa, which shows apparent differentiation of short and long shoots, than in the other six species, which do not show such differentiation. In another species that shows no differentiation of short and long shoots, Clerodendron trichotomum, LDI increased linearly and gradually with SL. This suggests that its long current-year shoots are superior to its short current-year shoots for both space-acquisition and leaf-display, and that the structural variation in its current-year shoots is small. The diverse patterns in the relationships between SL and LDI of current-year shoots are related to the variations among current-year shoots in the mean leaf number per unit stem length and the mean individual leaf area. Received 31 July 1998/ Accepted in revised form 7 June 1999     

Genetic Similarity of Flag Shoot and Ascospore Subpopulations of Erysiphe necator in Italy

The overwintering mode of the grape powdery mildew fungus, Erysiphe necator (syn. Uncinula necator), as mycelium in dormant buds (resulting in symptoms known as flag shoots) or as ascospores in cleistothecia, affects the temporal dynamics of epidemics early in the growing season. We tested whether distinct genetic groups (I and III) identified previously in E. necator correlate to overwintering modes in two vineyards in Tuscany, Italy, to determine whether diagnostic genetic markers could be used to predict overwintering. Samples from one vineyard were collected from flag shoots; the other vineyard, 60 km away, had no flag shoots, and mildew colonies were assumed to be derived from ascospores. Genetic markers putatively diagnostic for groups I and III showed that both types were common in the flag shoot subpopulation. Both genetic types were found in the ascospore population, although group III was dominant. We did not find strong genetic differentiation between the two subpopulations based on inter-simple sequence repeat markers. Although there was significant (P < 0.001) genetic differentiation between these subpopulations in 1997 and when 1997 and 1998 subpopulations were pooled (θ = 0.214 and 0.150, respectively), no differentiation was evident between vineyards in 1998 (θ = 0.138, P = 0.872). Moreover, we did not observe distinct lineages corresponding to overwintering modes, as observed in previous studies. We could not determine if differentiation resulted from biological differences or restricted gene flow between the two vineyards. Our samples were taken from both subpopulations early in the epidemic, while previous studies confounded overwintering mode and sampling time. These results do not support a strong correlation between overwintering and genetic groups, highlighting the need to base population biology studies on sound biological and epidemiological knowledge.     

Laboratory and field studies supporting the development of Heringia calcarata as a candidate biological control agent for Eriosoma lanigerum in New Zealand

Studies supporting a project seeking to introduce Heringia calcarata (Loew) (Diptera: Syrphidae) to New Zealand (NZ) to supplement biological control of woolly apple aphid (WAA), Eriosoma lanigerum (Hausmann) (Hemiptera: Aphididae) are reported. Annual surveys of H. calcarata presence and abundance in a Virginia, USA apple orchard revealed a bimodal distribution, with peaks in mid-June and mid-September. In the field, female H. calcarata oviposited on sentinel apple shoots infested with WAA, providing an efficient method for egg collection and larval production. Similarly, most field-collected females readily deposited viable eggs on WAA colonies in laboratory cages, demonstrating that mated females will oviposit in captivity. Survivorship of eggs and larvae transported to NZ was good, yielding adult flies in containment in Auckland. Adult, virgin female H. calcarata reared from eggs in captivity developed mature oocytes, providing an important step toward future mating studies in containment. Oviposition and larval feeding studies examined aspects of the intraguild interactions between H. calcarata and Aphelinus mali (Haldeman) (Hymenoptera: Aphelinidae), the sole biological control agent of WAA in NZ. Field tests using paired sentinel apple shoots with a non-parasitized or parasitized WAA colony revealed that although H. calcarata deposited eggs on both parasitized and non-parasitized colonies, fewest eggs were deposited on heavily parasitized colonies. Feeding studies showed that larval H. calcarata consumed fewer mummified aphids or aphids in an earlier stage of parasitization than non-parasitized aphids.     

Genomewide analysis of <Emphasis Type="Italic">ABCB</Emphasis>s with a focus on <Emphasis Type="Italic">ABCB1</Emphasis> and <Emphasis Type="Italic">ABCB19</Emphasis> in <Emphasis Type="Italic">Malus domestica</Emphasis>

The B subfamily of ATP-binding cassette (ABC) proteins (ABCB) plays a vital role in auxin efflux. However, no systematic study has been done in apple. In this study, we performed genomewide identification and expression analyses of the ABCB family in Malus domestica for the first time. We identified a total of 25 apple ABCBs that were divided into three clusters based on the phylogenetic analysis. Most ABCBs within the same cluster demonstrated a similar exon–intron organization. Additionally, the digital expression profiles of ABCB genes shed light on their functional divergence. ABCB1 and ABCB19 are two well-studied auxin efflux carrier genes, and we found that their expression levels are higher in young shoots of M106 than in young shoots of M9. Since young shoots are the main source of auxin synthesis and auxin efflux involves in tree height control. This suggests that ABCB1 and ABCB19 may also take a part in the auxin efflux and tree height control in apple.     

Genetic diversity and structure of local apple cultivars from Northeastern Spain assessed by microsatellite markers

A set of 493 old and local Spanish accessions of apple (Malus x domestica Borkh) maintained at three collections in Northeastern Spain was studied using 16 simple sequence repeats in order to estimate their genetic diversity and to identify the genetic structure and relationships among their accessions. An additional diverse set of 45 apple cultivars, including old Spanish and international cultivars, was added as reference. Genetic analyses performed by Bayesian model-based clustering revealed a very strong differentiation of two major groups. The first one clustered 159 individuals (52?% of unique genotypes) including local accessions and six old Spanish cultivars. The second major group was formed by 145 individuals, including 38 international reference cultivars and one old Spanish cultivar. Nested Bayesian clustering was applied to those two groups and two and four sub-groups were found at each one, respectively. The identification of private and unique alleles, and the remarkable differences in allelic richness among groups and sub-groups constitute further evidence of a clear genetic structure. The results obtained through the factorial correspondence and analyses of molecular variance confirmed those obtained by Bayesian analyses, revealing moderate but significant differentiation among the two major groups (F _ST?=?0.076) and the six sub-groups (F _ST?=?0.111). Our results highlight that the genetic diversity encompassed by currently cultivated apple accounts only for a small fraction of that existing within the species, and that an important part (??60?%) of the local material analyzed constitutes a good example of genetic distinctness with respect to the main cultivars used in European orchards.     

Evidence from the domestication of apple for the maintenance of autumn colours by coevolution

The adaptive value of autumn colours is still a puzzle for evolutionary biology. It has been suggested that autumn colours are a warning signal to insects that use the trees as a host. I show that aphids (Dysaphis plantaginea) avoid apple trees (Malus pumila) with red leaves in autumn and that their fitness in spring is lower on these trees, which suggests that red leaves are an honest signal of the quality of the tree as a host. Autumn colours are common in wild populations but not among cultivated apple varieties, which are no longer under natural selection against insects. I show that autumn colours remain only in the varieties that are very susceptible to the effects of a common insect-borne disease, fire blight, and therefore are more in need of avoiding insects. Moreover, varieties with red leaves have smaller fruits, which shows that they have been under less effective artificial selection. This suggests a possible trade off between fruit size, leaf colour and resistance to parasites. These results are consistent with the hypothesis that autumn colours are a warning signal to insects, but not with other hypotheses.     

Formation of biphenyl and dibenzofuran phytoalexins in the transition zones of fire blight-infected stems of Malus domestica cv. ‘Holsteiner Cox’ and Pyrus communis cv. ‘Conference’

In the rosaceous subtribe Pyrinae (formerly subfamily Maloideae), pathogen attack leads to formation of biphenyls and dibenzofurans. Accumulation of these phytoalexins was studied in greenhouse-grown grafted shoots of Malus domestica cv. ‘Holsteiner Cox’ and Pyrus communis cv. ‘Conference’ after inoculation with the fire blight bacterium, Erwinia amylovora. No phytoalexins were found in leaves. However, both classes of defence compounds were detected in the transition zone of stems. The flanking stem segments above and below this zone, which were necrotic and healthy, respectively, were devoid of detectable phytoalexins. The transition zone of apple stems contained the biphenyls 3-hydroxy-5-methoxyaucuparin, aucuparin, noraucuparin and 2′-hydroxyaucuparin and the dibenzofurans eriobofuran and noreriobofuran. In pear, aucuparin, 2′-hydroxyaucuparin, noreriobofuran and in addition 3,4,5-trimethoxybiphenyl were detected. The total phytoalexin content in the transition zone of pear was 25 times lower than that in apple. Leaves and stems of mock-inoculated apple and pear shoots lacked phytoalexins. A number of biphenyls and dibenzofurans were tested for their in vitro antibacterial activity against some Erwinia amylovora strains. The most efficient compound was 3,5-dihydroxybiphenyl (MIC = 115 μg/ml), the immediate product of biphenyl synthase which initiates phytoalexin biosynthesis.     

Winter peridermal conductance of apple trees: lammas shoots and spring shoots compared

Lammas shoots are flushes formed by some woody species later in the growing season. Having less time to develop, tissue formation is suggested to be incomplete leading to a higher peridermal water loss during consecutive months. In this study, we analysed morphological and anatomical parameters, peridermal conductance to water vapour and the level of native embolism in mid-winter and late-winter of lammas shoots and normal spring shoots of the apple varieties Malus domestica ‘Gala’ and ‘Nicoter’. Lammas shoots showed a significantly higher shoot cross-sectional area due to larger pith and corticular parenchyma areas. In contrast, phloem was significantly thicker in spring shoots. No pronounced differences were observed in xylem and collenchyma thickness or mean hydraulic conduit diameter. The phellem of spring shoots was composed of more suberinised cells compared to lammas shoots, which led to a significantly higher peridermal conductance in the latter. The amount of native embolism in mid-winter did not differ between shoot types, but in late-winter lammas shoots were more embolised than spring shoots. Data show that the restricted vegetation period of lammas shoots affects their development and, in consequence, their transpiration shield. This may also pose a risk for winter desiccation.     

Temporal requirement for phytohormone balance in the control of organogenesis in vitro

Leaf explants of Convolvulus arvensis produce roots or shoots when cultured in vitro on media of appropriate phytohormone balance. Each of these processes of organogenesis can be divided into three parts: phase 1, the acquisition of competence for induction, phase 2, induction per se, and phase 3, morphological differentiation and growth. Control of the type of organogenesis by the balance of exogenous phytohormones resides in phase 2; phases 1 and 3 occur over a wide range of hormone balances. The competence for induction acquired in phase 1 has a positional component. Root-, shoot-, and callus-inducing media, RIM, SIM, or CIM, respectively, all can produce competence for both root and shoot induction; however, roots arise from cells in the upper part of the callus, and shoots, from cells in contact with the medium. Some genotypes of Convolvulus do not make roots on RIM, others, make no shoots on SIM. Because explants of some of these genotypes can be made to regenerate organ types by short precultures on seemingly inappropriate media, we conclude that these genotypes are blocked in the acquisition of competence.     

A novel plasmid pEA68 of Erwinia amylovora and the description of a new family of plasmids

Recent genome analysis of Erwinia amylovora, the causal agent of fire blight disease on Rosaceae, has shown that the chromosome is highly conserved among strains and that plasmids are the principal source of genomic diversity. A new circular plasmid, pEA68, was found in E. amylovora strain 692 (LMG 28361), isolated in Poland from Sorbus (mountain ash) with fire blight symptoms. Annotation of the 68,763-bp IncFIIa-type plasmid revealed that it contains 79 predicted CDS, among which two operons (tra, pil) are associated with mobility. The plasmid is maintained stably in E. amylovora and does not possess genes associated with antibiotic resistance or known virulence genes. Curing E. amylovora strain 692 of pEA68 did not influence its virulence in apple shoots nor amylovoran synthesis. Of 488 strains of E. amylovora from seventeen countries, pEA68 was only found in two additional strains from Belgium. Although the spread of pEA68 is currently limited to Europe, pEA68 comprises, together with pEA72 and pEA78 both found in North America, a new plasmid family that spans two continents.     

Genomic Evidence for Speciation with Gene Flow in Broadcast Spawning Marine Invertebrates

How early stages of speciation in free-spawning marine invertebrates proceed is poorly understood. The Western Pacific abalones, Haliotis discus, H. madaka, and H. gigantea, occur in sympatry with shared breeding season and are capable of producing viable F₁ hybrids in spite of being ecologically differentiated. Population genomic analyses revealed that although the three species are genetically distinct, there is evidence for historical and ongoing gene flow among these species. Evidence from demographic modeling suggests that reproductive isolation among the three species started to build in allopatry and has proceeded with gene flow, possibly driven by ecological selection. We identified 27 differentiation islands between the closely related H. discus and H. madaka characterized by high F_ST and d_A, but not high d_XY values, as well as high genetic diversity in one H. madaka population. These genomic signatures suggest differentiation driven by recent ecological divergent selection in presence of gene flow outside of the genomic islands of differentiation. The differentiation islands showed low polymorphism in H. gigantea, and both high F_ST, d_XY, and d_A values between H. discus and H. gigantea, as well as between H. madaka and H. gigantea. Collectively, the Western Pacific abalones appear to occupy the early stages speciation continuum, and the differentiation islands associated with ecological divergence among the abalones do not appear to have acted as barrier loci to gene flow in the younger divergences but appear to do so in older divergences.     

Cytokinin-induced changes in the chlorophyll content and fluorescence of in vitro apple leaves

Cytokinins (CKs) are one of the main regulators of in vitro growth and development and might affect the developmental state and function of the photosynthetic apparatus of in vitro shoots. Effects of different cytokinin regimes including different types of aromatic cytokinins, such as benzyl-adenine, benzyl-adenine riboside and 3-hydroxy-benzyladenine alone or in combination were studied on the capacity of the photosynthetic apparatus and the pigment content of in vitro apple leaves after 3 weeks of culture. We found that the type of cytokinins affected both chlorophyll a and b contents and its ratio. Chlorophyll content of in vitro apple leaves was the highest when benzyl-adenine was applied as a single source of cytokinin in the medium (1846–2176 μg/1 g fresh weight (FW) of the leaf). Increasing the concentration of benzyl-adenine riboside significantly decreased the chlorophyll content of the leaves (from 1923 to 1183 μg/1 g FW). The highest chl a/chl b ratio was detected after application of meta-topolin (TOP) at concentrations of 2.0 and 6.0 μM (2.706 and 2.804). Chlorophyll fluorescence was measured both in dark-adapted (F_v/F_m test) and in light-adapted leaf samples (Yield test; Y(II)). The maximum quantum yield and efficiency of leaves depended on the cytokinin source of the medium varied between 0.683 and 0.861 (F_v/F_m) indicating a well-developed and functional photosynthetic apparatus. Our results indicate that the type and concentration of aromatic cytokinins applied in the medium affect the chlorophyll content of the leaves in in vitro apple shoots. Performance of the photosynthetic apparatus measured by chlorophyll fluorescence in the leaves was also modified by the cytokinin supply. This is the first ever study on the relationship between the cytokinin supply and the functionability of photosystem II in plant tissue culture and our findings might help to increase plantlet survival after transfer to ex vitro conditions.     

Thermal biology of Phymaturus lizards: evolutionary constraints or lack of environmental variation?

Several aspects of the biology of Phymaturus lizards including their herbivorous diet, specialized microhabitat use, and viviparous reproductive mode are highly conserved within the group. Here, we explore two aspects of Phymaturus thermal biology and test for the co-evolution among aspects of the thermal biology in these lizards, such as thermal preferenda and critical temperatures. Secondly, we explore correlations among variation in thermal biology with elevation and latitude. To do so, we used phylogenetically based comparative analyses (PCM) together with conventional statistics. Our results show that thermal biology for Phymaturus is conservative and our data do not suggest the co-evolution of thermal variables. Moreover, we detected low levels of variation in the thermal parameters studied, and no clear relationships between climatic and thermal variables. As a significant association between climatic and thermal variables could be demonstrated for a set of syntopic Liolaemus lizards, we suggest that thermal biology in Phymaturus lizards may be evolutionarily or ecologically constrained.