In vitro germination and shoot proliferation of the threatened species Cinchona officinalis L(Rubiaceae)

Cinchona officinalis(Rubiaceae) is an endemic species of the Loja Valley in southern Ecuador with medicinal uses. Because of over-exploitation in the nineteenth century and more recent disturbances to its ecosystem, C. officinalis populations are threatened. Currently,natural regeneration of the populations is low, despite its high plant regeneration and seed formation capacity. In the present study, an efficient protocol for germination, shoot proliferation and plantlets regeneration was developed for this species. Phenolic content and germination rate of C.officinalis seeds were compared with a control species, C.pubescens. Nodal segments from seedlings of C. officinalis were cultured on Gamborg medium supplemented with different combinations of plant growth regulators. Because the phenol content is high in C. officinalis, the phenolic should be removed with hydrogen peroxide or water washes to stimulate germination. Shoots and callus developed from nodal segments within 45 days using most of the tested combinations of plant growth regulators. The best rates of shoot proliferation, callus formation and adventitious buds were obtained in medium supplemented with 5.0 mg L~(-1) 6-benzyl-aminopurine and 3.0 mg L~(-1) indole-3-butyric acid.     

Genetic diversity in two threatened species in Vietnam: <Emphasis Type="Italic">Taxus chinensis</Emphasis> and <Emphasis Type="Italic">Taxus wallichiana</Emphasis>

Taxus chinensis and T. wallichiana in have been threatened in their distribution areas in recent decades because of their over-exploitation and reduction and destruction of native habitats. Determining the genetic diversity in populations of the two species will provide guidelines for their protection and preservation. Two hundred and fifteen trees from six populations of T. chinensis and 150 sampled trees of T. wallichiana were sampled. Six microsatellite primer pairs selected from 16 primer pairs were used to investigate genetic variation at the population and species levels. Five yielded polymorphic alleles, and among the 13 putative alleles amplified, 11 were polymorphic (accounting for 76.33 %).Shannon’s information index (I) and percentage of polymorphic bands (PPB) (I = 0.202 and PPB = 67.22 % for T. chinensis; I = 0.217 and PPB = 65.03 % for T. wallichiana). Both species had low levels of genetic diversity (mean H _o = 0.107, H _e = 0.121 for T. chinensis; H _o = 0.095, H _e = 0.109 for T. wallichiana). Genetic differentiation among populations was higher (F _ST = 0.189) for T. chinensis and lower (0.156) for T. wallichiana, indicating limited gene flow (Nm) among populations for T. chinensis (0.68) and T. wallichiana (0.65). Variation among individuals of T. chinensis was 63.59 and 73.12 % for T. wallichiana. Thus, the threatened status of the two conifers is related to a lack of genetic diversity. All populations are isolated in small forest remnants. An ex situ conservation site should be established with a new population for these species that comprises all the genetic groups for the best chance to improve their fitness under environmental stresses.     

In vitro clonal propagation of <Emphasis Type="Italic">Balanites aegyptiaca</Emphasis> (L.) Del

In vitro propagation technique of Balanites aegyptiaca, a multipurpose woody tree was studied. Nodal segments including axillary bud from mature tree were used as an explant and their morphogenetic potential was tested on MS media with various concentrations (2.5–15.0 μM) of 6-benzyladenine (BA), Kinetin, and Thidiazuron alone or in combination with different concentrations (0.5–2.5 μM) of α-naphthalene acetic acid (NAA). Nodal segments showed axillary bud proliferation in almost all media tried. MS medium containing 12.5 μM BA alone was effective for inducing multiple shoots (5.0 ± 0.22) with an average shoot length (3.7 ± 0.26 cm) in 67% of cultures. A better shoot differentiation and elongation was achieved in a combined treatment of BA (12.5 μM) and NAA (1.0 μM). Half strength MS medium supplemented with Indole-3-butyric acid (IBA) gave the best result for rooting. The maximum frequency of root formation (68%), number of roots (5.3 ± 0.32) and root length (4.1 ± 0.38 cm) was obtained on half strength MS medium containing 1.0 μM IBA. The regenerated plantlets were potted and acclimatized successfully in a growth chamber and then moved to the greenhouse.     

Antibacterial activity of moso bamboo shoot skin (<Emphasis Type="Italic">Phyllostachys pubescens</Emphasis>) against <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

This study examined the antibacterial activity of moso bamboo shoot skin (Phyllostachys pubescens). Bamboo shoot skin itself and its dichloromethane extract had antibacterial activity against Staphylococcus aureus. Results suggest the possibility of effective utilization of antibacterial materials from bamboo shoot skins that are mainly discarded at present.     

Successful <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Populus tomentosa</Emphasis> with apple <Emphasis Type="Italic">SPDS</Emphasis> gene

The problem of salinized soils has become one of the most serious constraints to agricultural and forest productivity. With the purpose of enhancing salt stress tolerance of Populus tomentosa, we transformed this tree species with spermidine synthase （SPDS） genes derived from an apple by an Agrobacterium-mediated method. Four transgenic clones were confu＇med by PCR and Southern blot analysis. As well, the expression of introduced SPDS genes was analyzed by real-time quantitative PCR.     

Pathogenic effects of <Emphasis Type="Italic">Xenorhabdus nematophilus</Emphasis> and <Emphasis Type="Italic">Photorhabdus luminescens</Emphasis> (Enterobacteriaceae) against pupae of the Diamondback Moth, <Emphasis Type="Italic">Plutella xylostella</Emphasis> (L.)

The relationship between Xenorhabdus nematophilus and Photorhabdus luminescens, the bacterial symbionts of the entomopathogenic nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora, against the diamondback moth, P. xylostella (L.), pupae showed the pathogenic capability of P. luminescens to be over that of X. nematophilus. They gave 60 and 40?% mortality with LC₅₀ values of 5?×?10⁴ and 5.5?×?10⁵ cells/ml, respectively.The number of bacterial cells influences the rate of killing the pupae of P. xylostella and a direct correlation between mortality of the pupae, percentage of deformed adults and the spraying dose with significant differences were observed. An inverse correlation found between the no. of eggs laid/female, percentage of hatching, adult survival and the spraying dose. These bacterial effects have been attributed to different toxic bacterial enzymes which damage the haemocytes and inhibit activation of the humoral immune system of the insect.     

Spatial pattern of beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.) and oak (<Emphasis Type="Italic">Quercus pubescens</Emphasis> Mill.) seedlings in natural pine (<Emphasis Type="Italic">Pinus sylvestris</Emphasis> L.) woodlands

Spatial pattern of recruitment is an important factor influencing population dynamics of plant communities. The spatial pattern is determined by seed dispersal and by the spatial variability of germination and initial survival. In the process of forest expansion following farmland abandonment, mid- and late-successional species are often dispersed in pioneer forests by birds. This could result in an aggregated spatial pattern of seeds that could influence the dynamics of these species in mixed pioneer forests. In the sub-Mediterranean area, mid- and late-successional species such as Quercus pubescens (downy oak) and Fagus sylvatica (European beech) are expected to replace pioneer Pinus species. Using a point sampling method we demonstrated that beech and oak seedlings (height <2 m) have a clumped distribution in the understorey of pine. This could result from an aggregated dispersal by jays (dispersal effect) or from preferential recruitment in particular habitats (habitat effect). To test these hypotheses we proposed a statistical analysis of spatial patterns of regeneration of beech and oak. Ground cover variables (i.e. cover by rock outcrops, herbs, box shrubs, mosses or pine) did not differ significantly around seedlings as compared with random sample plots. Likewise, clumped seedlings had growth similar to isolated seedlings, thus refuting the hypothesis of preferential location in the most favourable microsites. Aggregated dispersal seems to be involved in the process of regeneration. Since beech and oak seedlings have contrasting ecological demands, we discuss the implication of this pattern for the replacement dynamics of pine by these species.     

Effect of different pre-sowing seed treatments on the germination of <Emphasis Type="Italic">Leucaena leucocephala</Emphasis> (Lam.) and <Emphasis Type="Italic">Acacia farnesiana</Emphasis> (L.)

Leucaena leucocephala and Acacia farnesiana are tree species used for several agricultural purposes in the Mediterranean region. The seeds of these species exhibit dormancy, causing delayed germination. Two experiments were conducted to investigate the effect of pre-sowing treatments (scarification, hot water, or soaking) on seed germination of L. leucocephala and A. farnesiana. In one experiment, seeds were exposed to three pre-sowing treatments: control, sandpaper scarification, or soaking in 70°C water for 4, 8, 12, 16, 20, or 24 min. In another experiment, seeds were soaked in 70°C water for 20 min, and then soaked in water at room temperature for an additional 24, 48, or 72 h or blade scarified. In general, soaking the seeds of the two species in hot water was more effective in breaking seed dormancy than scarification. Sandpaper scarification was not effective for either species. Blade scarification increased A. farnesiana seed germination to 56%, indicating that seed dormancy was mainly a consequence of hardseededness. L. leucocephala seeds collected from Jordan University of Science and Technology (JUST) site and soaked in 70°C water for 20 min and then soaked for 24, 48, or 72 h had germination rates above 97%. Our results suggest that blade scarification of A. farnesiana seeds and soaking of L. leucocephala seeds in 70°C water for 20 min are effective treatments to break seed dormancy and enhance seed germination of these vital species.     

Parasitism of different <Emphasis Type="Italic">Trichogramma</Emphasis> species and strains on <Emphasis Type="Italic">Plutella xylostella</Emphasis> L. on greenhouse cauliflower

The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is the most important pest of cultivated Brassica worldwide, including those grown in greenhouses like cauliflower. In this article, we evaluate the potential of various species (and various strains of some species) of Trichogrammatidae (Hymenoptera: Chalcidoidea) to control this pest on cauliflower in greenhouse in France. We assessed the parasitism levels on P. xylostella of 17 Trichogrammatidae strains, belonging to 12 different species (2 indigenous strains from France), under greenhouse conditions. Parasitism levels for each of the Trichogrammatidae species and strains were determined on cauliflower leaves (Brassica oleracea botrytis L., Brassicaceae) infested with P. xylostella eggs. Nine strains parasitized 60% (or more) of the P. xylostella eggs. Compared to previous results in laboratory conditions, climatic conditions of the greenhouse did not influence parasitism levels. The presence of the cauliflower plants may have a positive effect on eight strains, a negative effect on four strains and no effect on five strains. Our study points out the importance of including the host plant of P. xylostella when conducting studies aiming to select the most efficient parasitoid against this pest.     

Insecticidal properties of <Emphasis Type="Italic">Thymus persicus</Emphasis> essential oil against <Emphasis Type="Italic">Tribolium castaneum</Emphasis> and <Emphasis Type="Italic">Sitophilus oryzae</Emphasis>

Red flour beetle Tribolium castaneum (Herbst) and rice weevil Sitophilus oryzae (L.) are considered to be the major insect pests in storage. Essential oils from aromatic plants are recognized as proper alternatives to fumigants. Thymus persicus (Ronniger ex Rech. f.) is one of these plants that have medicinal properties and is indigenous to Iran. The essential oil was obtained from aerial parts of the plant and analyzed by GC and GC–MS. Carvacrol (44.69%) and thymol (11.05%) were the major constituents of the oil extracted. In this experiment, fumigant toxicity of the essential oil was studied against T. castaneum, S. oryzae at 27 ± 1°C and 60 ± 5% RH in dark condition. The adult insects were exposed to the concentrations of 51.9, 111.1, 207.4 and 370.4 μl/l air to estimate median lethal time (LT₅₀) values. The fumigant toxicity was increased in response to increased essential oil concentrations. The LT₅₀ values at the lowest and the highest concentrations tested were ranged from 28.09 to 13.47 h for T. castaneum, and 3.86 to 2.30 h for S. oryzae. It was found that S. oryzae adults were much more susceptible to the oil than T. castaneum. After 24 h of exposure, the LC₅₀ values (95% fiducial limit) for T. castaneum and S. oryzae were estimated to be 236.9 (186.27–292.81) and 3.34 (2.62–4.28) μl/l air, respectively. These results suggest that T. persicus essential oil merits further study as potential fumigant for the management of these stored-product insects.     

<Emphasis Type="Italic">In vitro</Emphasis> morphogenic response of leaf sheath of <Emphasis Type="Italic">Phyllostachys bambusoides</Emphasis>

Nodal segments from secondary branches of saplings of Phyllostachys bambusoides were inoculated in MS medium to assess the in vitro morphogenic response of leaf sheath through the induction to callogenesis by Picloram(4-amino-3,5,6-trichloropicolinic acid) at different concentrations of carbohydrate under the same conditions with presence or absence of luminosity.In our experiment,secondary explants were kept in MS medium containing 8.0 mg·L-1 of Picloram for the callus formation.Calluses were transferred in MS medium supplemented with sucrose,fructose and glucose(control,2%,4% and 6%).Results show that Picloram induced the callogenesis in leaf sheath.The secondary embryogenesis was formed in yellow-globular callus.The sucrose as carbohydrate source in the absence of light was more efficient to induce rhizogenesis.Glucose was more efficiency in the presence of light.Callogenic induction and further embryogenesis evidenced the competence and determination of leaf sheath cells.     

In vitro regeneration of <Emphasis Type="Italic">Populus tomentosa</Emphasis> from petioles

A reliable in vitro regeneration procedure for Populus tomentosa is a prerequisite for its trait improvement through genetic transformation. We established a systematic protocol for indirect regeneration of P. tomentosa using in vitro petioles of Chinese poplar cultivar ‘fasta-3’. A high frequency of callus induction (>97 %) was obtained from isolated petioles cultured on the modified 1/2MS basal medium supplemented with 0.5 mg/L ZT and 1.0 mg/L NAA, and the tested calli were subsequently plated on 1/2MS basal medium supplemented with 0.25 mg/L BA, 0.25 mg/L ZT, 0.25 mg/L NAA, 0.01 mg/L TDZ, and 0.5 mg/L KT for efficient regeneration of shoots after being cultured for 6 weeks. The regenerated shoots were vigorously rooted on the tested media supplemented with 1.0 mg/L IBA and 0.5 mg/L NAA. These results can facilitate genetic transformation of P. tomentosa for trait improvements in future.     

Direct and admixture toxicity of diatomaceous earth and monoterpenoids against the storage pests <Emphasis Type="Italic">Callosobruchus</Emphasis><Emphasis Type="Italic">maculatus</Emphasis> (F.) and <Emphasis Type="Italic">Sitophilus oryzae</Emphasis> (L.)

We investigated the effects of two commercial diatomaceous earth based insecticides (DE), Protect-It^® and SilicoSec^®, the nano-structured silica product AL06, developed by the section for Urban Plant Ecophysiology at Humboldt University Berlin, and the monoterpenoids, eugenol, and cinnamaldehyde on two stored product pests, Callosobruchus maculatus and Sitophilus oryzae. Protect-It^® was more effective than SilicoSec^® against C. maculatus while the reverse was true for S. oryzae. Generally C. maculatus was more sensitive towards DE and silica treatment than S. oryzae. Mortality rate of both pest species increased when DE’s were applied to food commodities previously treated with a monoterpenoid. In admixture experiments, the toxicity of SilicoSec^® + cinnamaldehyde (LD₅₀ = 42.73 ppm), SilicoSec^® + eugenol (LD₅₀ = 24.30 ppm), and Protect-It^® + eugenol (LD₅₀ = 2.60 ppm) was increased over DE alone against S. oryzae. Both substances showed a synergistic effect considering their co-toxicity coefficient relative to the LD₅₀-value. In contrast, we could not find any synergistic effects in experiments with C. maculatus. Here only Protect-It^® + cinnamaldehyde (LD₅₀ = 20.84 ppm) showed an additive effect while all other combinations of monoterpenoid and DE indicated antagonistic effects. In addition to contact insecticidal effects both monoterpenoids showed a strong fumigant action. The presented results indicate that the natural product DE has great potential to replace synthetic pesticides commonly used in stored product pest management. Efficacy of DE can be improved by adding certain monoterpenoids against certain insect pests.     

Allelopathic effects of <Emphasis Type="Italic">Pinus halepensis</Emphasis> and <Emphasis Type="Italic">Quercus coccifera</Emphasis> on the germination of Mediterranean crop seeds

Agroforestry is a leading alternative for food security and forest conservation. A full understanding of positive and negative, i.e. allelopathic, interactions between crops and forest trees is necessary for producing crops and conserving forests especially within the threatened Mediterranean forest ecosystems. The present study explored the allelopathic effects of green and senescent leaf and soil extracts of two agroforestry trees—Pinus halepensis and Quercus coccifera—on the germination of wheat, barley, lentil, chickpea, and fababean as the major grain crops of Jordan. Results revealed that allelopathic effects reduced seed germination of these crops. Germination reduction reached a maximum of 75% in fababean treated with green extracts of Q. coccifera and differed among crops and extract sources, but not between tree species. Comparing between green and senescent leaf and soil extract, regarding their effect on germination percentage, it was noticed that these effects were similar in some crops and were different in others. Germination responses were generally different between cereals and legumes where cereals tend to be less affected by allelopathic influences than legumes, especially fababean. We suggest using cereals such as wheat and barley in agroforestry practices in the Mediterranean region of Jordan.     

<Emphasis Type="Italic">Ophiostoma</Emphasis> species associated with bark beetles infesting three <Emphasis Type="Italic">Abies</Emphasis> species in Nikko,Japan

Ophiostoma species were isolated from bark beetles and Abies mariesii, A. veitchii and A. homolepis attacked by the beetles in Nikko, Tochigi, central Honshu, Japan. One to two Ophiostoma species were frequently isolated from each species of bark beetle. Ophiostoma subalpinum was the most common associate of Cryphalus montanus. Ophiostoma sp. B as well as O. subalpinum was a common fungus associated with Polygraphus proximus. Ophiostoma europhioides was isolated from Dryocoetes hectographus and D. autographus as one of the common associates. Ophiostoma sp. J and Ophiostoma sp. S were frequently isolated from D. autographus and D. striatus, respectively. These fungi seem to have specific relationships with particular bark beetles. Ophiostoma sp. B, Ophiostoma sp. J and Ophiostoma sp. S have unique morphological characteristics and appear to be new species. Five trees of A. veitchii, approximately 43 years old, were inoculated with five Ophiostoma species to assess the relative virulence of the fungi. Ophiostoma subalpinum, Ophiostoma sp. B, and O. europhioides had relatively higher virulence than the other species studied.     

In vitro propagation of the African mahogany <Emphasis Type="Italic">Khaya senegalensis</Emphasis>

A protocol was developed for shoot proliferation and plantlet formation of Khaya senegalensis, an important medicinal and timber plantation species introduced to Australia and southern Asia from western and central Africa. We assessed effects of the plant growth regulators, benzyladenine, kinetin, naphthalene acetic acid and gibberellic acid, on shoot proliferation and subsequent plantlet conversion. Shoot proliferation over four passages was higher in media containing benzyladenine than in media containing other growth regulators, and optimal proliferation from seed of three different sources was consistently obtained in medium containing 4.4 μM benzyladenine. Shoots from this medium were converted to plantlets at high frequencies (76–90%) after treatment with 19.6 μM indole-3-butyric acid, and almost all plantlets were successfully acclimatized to nursery conditions. These methods provide the means for establishing in vitro and ex vitro clone banks of juvenile K. senegalensis trees for field selection of desired genotypes and tropical plantation establishment.     

Effectiveness of Neem (<Emphasis Type="Italic">Azadirachta indica</Emphasis>) insecticides against Brassica pod midge (<Emphasis Type="Italic">Dasineura brassicae</Emphasis> Winn<Emphasis Type="Italic">.</Emphasis>)

The efficiency of the botanical insecticide (BI) NeemAzal T/S (containing 1% Azadirachtin A), on the basis of azadirachtin applied in a dose of 20 g a.i. ha⁻¹ against Brassica pod midge (Dasineura brassicae), has been monitored for 4 years. The biological efficiency of BI was compared with the efficiency of some synthetic insecticides. It was ascertained that BI was very efficient in decreasing the number of damaged oilseed rape pods (ranging from 56.5 to 85.9% compared to untreated plants) and its efficiency was comparable with synthetic insecticides based on Chloronicotinyl (Thiacloprid) and Neonicotinoid (Acetamiprid). BI’s efficiency was, in some years, even significantly higher compared to pyrethroid (λ-cyhalothrin). The high biological efficiency of azadirachtin that we ascertained was significantly reflected in increasing potential crop yields. The yield increase of azadirachtin ranged between 9.3 and 19.4% compared to the control sample. Azadirachtin showed the highest yield for the whole time of experimentation, and in some years the yield increase was significantly higher compared to some synthetic agents.     

Bioremediation of CCA-treated wood by brown-rot fungi <Emphasis Type="Italic">Fomitopsis palustris</Emphasis>, <Emphasis Type="Italic">Coniophora puteana</Emphasis>, and <Emphasis Type="Italic">Laetiporus sulphureus</Emphasis>

This study evaluated oxalic acid accumulation and bioremediation of chromated copper arsenate (CCA)-treated wood by three brown-rot fungi Fomitopsis palustris, Coniophora puteana, and Laetiporus sulphureus. The fungi were first cultivated in a fermentation broth to accumulate oxalic acid. Bioremediation of CCA-treated wood was then carried out by leaching of heavy metals with oxalic acid over a 10-day fermentation period. Higher amounts of oxalic acid were produced by F. palustris and L. sulphureus compared with C. puteana. After 10-day fermentation, oxalic acid accumulation reached 4.2 g/l and 3.2 g/l for these fungi, respectively. Fomitopsis palustris and L. sulphureus exposed to CCA-treated sawdust for 10 days showed a decrease in arsenic of 100% and 85%, respectively; however, C. puteana remediation removed only 18% arsenic from CCA-treated sawdust. Likewise, chromium removal in F. palustris and L. sulphureus remediation processes was higher than those for C. puteana. This was attributed to low oxalic acid accumulation. These results suggest that F. palustris and L. sulphureus remediation processes can remove inorganic metal compounds via oxalic acid production by increasing the acidity of the substrate and increasing the solubility of the metals.An erratum to this article can be found at     

Enzymatic saccharification and ethanol production of <Emphasis Type="Italic">Acacia mangium</Emphasis> and <Emphasis Type="Italic">Paraserianthes falcataria</Emphasis> wood,and <Emphasis Type="Italic">Elaeis guineensis</Emphasis> trunk

We examined the saccharification and fermentation of meals from Acacia mangium wood, Paraserianthes falcataria wood, and Elaeis guineensis trunk. The levels of enzymatic hydrolysis of cellulose and ethanol production were highest for P. falcataria wood and lowest for A. mangium wood. Ultrasonication pretreatment of meal further increased the rates of hydrolysis and ethanol production in meal from P. falcataria wood. Through this pretreatment, hemicelluloses (xylan and xyloglucan) and cellulose were released in the meal from P. falcataria wood. Loosening of hemicellulose associations can be expected to make P. falcataria wood more useful for bioethanol production.