Flow cytometric analysis and molecular characterization of Agrobacterium tumefaciens-mediated transformants of Medicago truncatula

Leaf explants from leaflets collected from either in vivo grown or in vitro grown seedlings of Medicago truncatula genotype R108-1 were co-cultivated with bacterial cells of Agrobacterium tumefaciens strains EHA105 or C58pMP90. Each of these strains was carrying the pCambia 1390 plasmid harbouring a hygromycin resistance gene cassette. Explants were then incubated on a medium containing 10 mg/l hygromycin and 800 mg/l augmentin to suppress Agrobacterium growth, and subcultured 4–5 times every 2 weeks for the proliferation of calli. After 8–10 weeks, callusing explants were transferred to hormone-free medium with 10 mg/l hygromycin and 400 mg/l augmentin for shoot regeneration. After rooting, a total of about 300 putative transformants were grown into plantlets, transferred to soil, acclimatized, and then moved to the greenhouse. Of these, a total of 43 independent PCR positive primary transformants and their T1 and T2 progeny were subjected to flow cytometric analysis, to assessing their trueness-to-type, as well as to southern blot analysis.     

Changes in predation and parasitism of the citrus leafminer Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae) populations in Spain following establishment of Citrostichus phyllocnistoides (Hymenoptera: Eulophidae)

The citrus leafminer, Phyllocnistis citrella, is a pest native to Southeast Asia which threatened the citrus industry in the Mediterranean region upon its introduction in 1993. Immediately afterward, a classical biological control program was implemented in Spain. The exotic parasitoid Citrostichus phyllocnistoides was the only introduced parasitoid to become established. In 2006, data on both the incidence of P. citrella and the impact of its natural enemies were collected following the same protocols used in 1997–1999 when C. phyllocnistoides was not yet present. C. phyllocnistoides constituted 99.4% of the parasitoids collected in 2006 corresponding to a decrease in the incidence of P. citrella from 3.2–5.1 to 1.8–2.4 mines per leaf in 1997–1999 and 2006, respectively. Mortality caused by natural enemies on P. citrella in 2006 was 93.3% (18.0% parasitism, 40.8% feeding punctures and 34.5% predation). C. phyllocnistoides, which preferentially parasitizes P. citrella second instar larvae, has displaced most of the indigenous parasitoids that moved onto P. citrella mainly parasitizing third instar larvae, upon its introduction. Because C. phyllocnistoides is an idiobiont parasitoid and preferentially parasitizes P. citrella second instars, this stage has become dominant in the orchards. The shift in the relative abundance of P. citrella larvae has prompted generalist predators to prey mostly on second instars and has contributed to the displacement of the native non-specific parasitoids, which principally utilize third instars, from the system. Both indigenous predators and the introduced parasitoid are key players in the natural regulation of P. citrella.     

Hydrogel substrate amendment alleviates drought effects on young citrus plants

Water deficits affect citrus physiology, yield, fruit size and quality. Citrus can respond to drought stress conditions through endogenous hormonal regulation of water status and leaf abscission. In this work, we assayed the efficiency of an amendment to soilless media in delaying the drought stress effect in young citrus seedlings and trees. Substrate amendment promoted plant survival of citrus seedlings subjected to several cycles of drought stress and rehydration. In budded trees, the amendment increased substrate water content, leaf water potential, leaf number, root biomass, CO₂ assimilation and stomatal conductance over that of control plants growing in non-amended substrates. We conclude that the substrate amendment reduced the damaging effects of drought stress in citrus plants. The longer survival of seedlings in the amended treatment together with the reduction in leaf abscission and the improvement of physiological parameters, can account for a higher vigour of citrus grown under water stress conditions.     

Isolation and characterization of polymorphic microsatellite markers in Tetranychus urticae and cross amplification in other Tetranychidae and Phytoseiidae species of economic importance

Tetranychus urticae Koch is a cosmopolitan phytophagous mite considered as the most polyphagous species among spider mites. Population genetic studies using molecular markers such as microsatellites have proven to be extremely informative to address questions about population structure, phylogeography and host preferences. The aim of this study was to increase the available molecular tools to gain insight into the genetic structure of T. urticae populations of citrus orchards, which might help in their management. Five microsatellite DNA libraries were developed using probes with the motifs CT, CTT, GT and CAC following the FIASCO protocol. Positive clones, those that included the insert with the microsatellite, were detected using the PIMA-PCR technique. Combinations of primers were designed on 22 out of 32 new microsatellites loci and their polymorphism was tested in four populations sampled along the eastern coast of Spain. Eleven successful amplifications were obtained. Cross amplification was tested in the tetranychids Aphlonobia histricina, Eutetranychus banksi, E. orientalis, Oligonychus perseae, Panonychus citri, Tetranychus evansi, T. okinawanus and T. turkestani, and the phytoseiids Amblyseius swirskii, A. cucumeris, A. andersoni, Euseius stipulatus, Neoseiulus barkeri, N. californicus, Phytoseiulus persimilis and Typhlodromus phialatus. Eight successful cross amplifications were obtained.     

Are the palms Washingtonia filifera and Chamaerops humilis suitable hosts for the red palm weevil,Rhynchophorus ferrugineus (Col. Curculionidae)?

The red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera, Curculionidae), is a widely distributed pest which has recently become the major pest of palms in the Mediterranean Basin. This weevil has been recorded on 19 different palm species, however, the host status of Washingtonia filifera and Chamaerops humilis remains unclear. The present study was carried out to check whether these species had either antixenotic or antibiotic mechanisms of resistance against this pest. Our results show that both W. filifera and C. humilis are resistant to R. ferrugineus. Resistance in W. filifera is based on antibiosis whereas in C. humilis resistance is based on antixenosis.     

Indigenous Natural Enemies Associated with Phyllocnistis citrella (Lepidoptera: Gracillariidae) in Eastern Spain

The incidence of generalist indigenous natural enemies of the citrus leafminer (CLM), Phyllocnistis citrella Stainton (Lepidoptera; Gracillariidae), was monitored during three growing seasons at two different orchards located in the major citrus-growing area of Spain. Composition of the parasitoid complex changed during the study period. However, the eulophids Cirrospilus near lyncus Walker and Pnigalio pectinicornis L. were consistently the predominant species. Despite the varying composition of the parasitoid complex, oviposition, host feeding, and predatory preferences of the natural enemies of the CLM clearly centered on third instar larvae. Incidence of beneficial fauna increased as the season progressed, reaching maximal values up to 70% of susceptible leafminers (mature larvae) at the end of the summer. Parasitism was significantly related to relative host density. However, predation showed no relationship to host availability but did so to flushing in one of the orchards. Incidence of indigenous natural enemies of the CLM should not be ignored when planning any introduction of exotic parasitoids, and their conservation should be taken into account when planning any citrus IPM strategy.     

Temperature Requirements may Explain why the Introduced Parasitoid Quadrastichus citrella Failed to Control Phyllocnistis citrella in Spain

Quadrastichus citrella Reina and La Salle (Hymenoptera: Eulophidae) is a parasitoid of Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae), indigenous to South Eastern Asia where it is considered a key natural enemy of this pest. It was introduced in Spain in a classical biological control program and became established, but was not as successful as forecast. To check whether its biology could explain this lack of control, development and reproduction at different temperatures were studied. Quadrastichus citrella can survive at temperatures between 15 and 35 °C. Lower development threshold was 12.3 °C, which resulted in a thermal constant of 138.06 DD. The combination of development and reproduction into demographic parameters resulted in highest net reproduction occurring in the range 20–25 °C (78.2 females per female), and highest intrinsic rate of increase at 30 °C (0.2571 females per female per day). These values make Q. citrella an intrinsically superior natural enemy compared to other leaf miner parasitoids. However, overwintering of Q. citrella in Spain may present a barrier, especially in areas like Valencia, where average winter temperatures are around 11 °C. This could account for the low recovery rates observed in Spain in spite of the key natural enemy status enjoyed by this species in its area of origin.     

Enzymatic and non-enzymatic antioxidant responses of Carrizo citrange,a salt-sensitive citrus rootstock,to different levels of salinity

In several plant species, oxidative stress has been shown to be one of the causes of damage produced by salinity. In order to assess the implication of oxidative stress in the reported sensitivity of the citrus rootstock Carrizo citrange to salt stress, 5-month-old seedlings were grown with increasing NaCl concentrations added to the watering solution. As an indicator of oxidative damage, malondialdehyde content was measured. The antioxidant capability of the plants was determined by measuring superoxide dismutase, ascorbate peroxidase, catalase and glutathione reductase activities together with the non-enzymatic antioxidant activity. As additional physiological responses to the stress, 1-aminocyclopropane-1-carboxilic acid and proline accumulation were assessed. Data indicate that Carrizo citrange responded to salt-induced oxidative stress by increasing enzymatic and non-enzymatic antioxidant defenses proportionally to the extent of the stress imposed, and that in all plants the malondialdehyde content remained at a moderate level. We suggest that the important deleterious effects reported in Carrizo citrange grown under high NaCl concentrations are mainly due to a cellular intoxication by Cl(-) ions and not to the salt-induced oxidative stress.     

Analysis of a laboratory method to test the effects of pesticides on adult females of Opius concolor (Hym., Braconidae), a parasitoid of the olive fruit fly,Bactrocera oleae (Dip., Tephritidae)

The laboratory test described in the present paper assesses pesticide effects on the ‘most exposed’ life stage of the parasitic wasp Opius concolor, and it has been developed and interpreted according to the guidelines of the IOBC/WPRS Working Group ‘Pesticides and Beneficial Organisms ‘. The evaluation of results is based on longevity and parasitism of adult females exposed to afresh residue of pesticide on a glass surface. Longevity measurements were based on the time of exposure required to cause 50% mortality (LT₅₀) and were compared to a standard control LT₅₀ in order to calculate the reduction caused by the pesticide. Parasitism was surveyed by means of three sequential determinations: the number of days that females were seen parasitizing, the mean number of attacked hosts and the progeny size per female. These determinations were also compared to a control to calculate the impact of pesticides. Using this ‘worst case’ situation, pesticides reducing the beneficial capacity of O. concolor below the IOBC threshold for harmlessness may be recommended for field use in combination with this wasp.     

Effect of Temperature on Development and Survival of Citrostichus phyllocnistoides (Hymenoptera: Eulophidae), a Parasitoid of Phyllocnistis citrella (Lepidoptera: Gracillariidae)

Thermal requirements of Citrostichus phyllocnistoides under laboratory conditions resulted in a lower temperature threshold of 9.8°C, and a thermal constant of 212.0 DD. Development was completed between 15 and 30°C, but was not at 10°C. Therefore, the overwintering of this exotic biocontrol agent would be possible under typical Mediterranean temperatures.