Micropropagation of mature <Emphasis Type="Italic">Pongamia pinnata</Emphasis> Pierre

Murashige and Skoog’s (MS) basal medium with benzylaminopurine (BA), kinetin (KN), zeatin (Z), and thidiazuron (TDZ) were tested for induction of multiple shoots from mature-tree-derived axillary meristems of Pongamia pinnata. Sprouting of buds was 64% on medium devoid of plant growth regulators (PGR). Incorporation of BA, KN, or Z was ineffective in enhancing sprouting frequency or induction of multiple shoots. Sprouting was completely suppressed in the presence of TDZ. Caulogenic buds appeared in nodal meristems of these explants after withdrawal of TDZ. The number of shoot buds was more on explants precultured in higher concentrations. At higher concentrations of this PGR, a swelling developed at the axil. Multiple shoot primordia appeared and differentiated from this swelling after culturing these explants on MS medium for six passages of 2 wk each. Shoots were harvested and cultured on 0.45 μM TDZ for further proliferation. Primary explants after harvesting of shoots were identified as ‘stump’. Reculturing of stumps on 0.45 μM TDZ produced more shoots. This step was followed for six cycles to obtain additional shoots in each cycle. Shoots maintained on 0.45 μM TDZ elongated and rooted (70%) on growth regulator-free medium. Rooted shoots (65%) survived transfer to a sand/soil mixture. This report describes the protocol for micropropagation of P. pinnata using mature-tree-derived nodal meristems. Recycling of mature stock to produce a stream of useable shoots for subculturing and eventual stabilization is of great value and can possibly be generalized as an isolation protocol especially for woody species. Repeated proliferation of caulogenic buds from the same origin may also find application in rescue of endangered germplasm.     

In vitro propagation of <Emphasis Type="Italic">Helleborus</Emphasis> species

A general in vitro cloning system was established for four Helleborus species: H. argutifolius, H. foetidus, H. niger and H. orientalis. The plant material was introduced in vitro from axillary buds. A Murashige and Skoog (MS)—based medium (Murashige and Skoog 1962) was used supplemented with 2% (w/v) sucrose, 2-isopentenyladenine (2-iP) and 6-benzylaminopurine (BA). Multiplication rates depended on the genotype and varied from 1.3 for H. foetidus till 3.8 for H. niger. The first results showed that the rooting phase could be done ex vitro. Rooting was induced by a drench for one week in a solution of indole-3-butyric acid (IBA -3 mg l⁻¹) and 1-naphthaleneacetic acid (NAA-1 mg l⁻¹) at 5°C.     

Decolourisation and dephenolisation potential of selected <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Nigri</Emphasis> strains – <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> in olive mill wastewater

Aspergillus section Nigri strains Aspergillus aculeatus Ege-K 258, A. foeditus var. pallidus Ege-K156, A. niger Ege-K 4 and A. tubingensis Ege-K 265 were used to treat olive mill wastewater (OMW) in an investigation aimed at exploring their dephenolisation and decolourisation ability and, consequently, the economic feasibility of using any or all of these strains in a pre-treatment step in the processing of OMW. Of these strains A. tubingensis Ege-K 265 resulted in an 80% decolourisation of twofold-diluted OMW and a 30% decolourisation of undiluted OMW; in addition, it was able to remove approximately 30% of all phenolic compounds in both twofold-diluted and undiluted OMW. We conclude that A. tubingensis Ege-K 265 could be effectively used in the pre-treatment step of a combined aerobic-anaerobic process to solve the environmental problems caused by OMW in Mediterranean countries.     

A new name in <Emphasis Type="Italic">Pavetta</Emphasis> L. (<Emphasis Type="Italic">Rubiaceae</Emphasis>)

Summary  The name Pavetta modesta (Hiern) S. E. Dawson is a later homonym of P. modesta Bremek. Pavetta crystalensis is proposed as a new name.     

Typification of <Emphasis Type="Italic">Metrosideros regelii</Emphasis> (<Emphasis Type="Italic">Myrtaceae</Emphasis>) and consideration of its generic position

Summary  The type specimen of Metrosideros regelii is discussed. It contains a mixture of two species, representing different genera, and a lectotype is chosen. The generic position of the species is considered in the light of morphology and recent molecular evidence and the new combination, Mearnsia regelii, made.     

<Emphasis Type="Italic">In vitro</Emphasis> selection and regeneration of chrysanthemum (<Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> Tzelev) plants resistant to culture filtrate of <Emphasis Type="Italic">Septoria obesa</Emphasis> Syd

Callus cultures derived from leaf segments of chrysanthemum cultivar ‘Snow Ball’ which was susceptible to Septoria obesa were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant cell lines were selected by culturing callus on growth medium containing various concentrations of S. obesa filtrate. Resistant calluses obtained after two cycles (30 d each cycle) of selection were used for plant regeneration. About 30% of the plants regenerated from the resistant calluses and 70–80% of the plants raised from cuttings had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

<Emphasis Type="Italic">Septobasidium parviflorae</Emphasis> sp. nov. on <Emphasis Type="Italic">Pinus parviflora</Emphasis> from Japan

Septobasidium parviflorae sp. nov. on Pinus parviflora is described and illustrated. This species is characterized by its whitish-gray, gray to dark gray-colored fungus body with an indeterminate margin, hyphal strands, and cylindrical basidia with long sterigmata. This is the first report of Septobasidium occurring on a member of the genus Pinus in Japan.     

cAMP promotes hyphal branching in <Emphasis Type="Italic">Mucor globosus</Emphasis>

The growth pattern of Mucor globosus cultured on a medium with or without cyclic adenosine monophosphate (cAMP) was examined. Branching remarkably increased in a mycelium grown on an agar medium containing cAMP. In submerged culture containing cAMP, some sporangiospores grew spherically and formed yeast-like cells, and others showed hyphal growth. These hyphae showed septation and swelling and formed spore-like structures. When these hyphae were transferred to cAMP-free medium, a germ tube emerged from each compartment. These results show that cAMP has two different effects on the development of hyphae: one is the promotion of branching, and the other is the suppression of polarized growth.     

Muscular architecture of <Emphasis Type="Italic">Milnesium tardigradum </Emphasis>and <Emphasis Type="Italic">Hypsibius </Emphasis>sp. (Eutardigrada,Tardigrada) with some data on <Emphasis Type="Italic">Ramazottius oberhaeuseri</Emphasis>

The architecture of the musculature of the eutardigrade species Milnesium tardigradum Doyère, 1840, Hypsibius sp. and Ramazzottius oberhaeuseri (Doyère in Ann Sci Nat Zool Sér 2(14):269–369, 1840) is investigated by phalloidin staining and confocal laser scanning microscopy. There are methodological problems in staining eutardigrades due to physiological alterations under stress (anhydrobiosis) and due to penetration problems of the cuticle. It is helpful to fix specimens in the state of asphyxy, where animals are stretched following an oxygen shortage in their environment. The musculatures of all three species correspond in their general architecture, but differ in detail, such as in the number of muscles. All muscles are isolated muscle strands. There are on each body side two dorsal and one ventral muscle strands, in addition to a system of dorsoventral, lateral and lateroventral muscles. Seven median ventral attachment points give rise to dorsoventral, ventrolateral and appendage muscles. The appendages receive several muscles originating dorsally and ventrally. The number of muscles and the arrangement differ in each appendage. The fourth appendage shows the greatest differences with a far smaller number of muscles compared to other species. The musculature shows comparably few strict segmental patterns, for example, the musculature of each appendage differs from the other ones. By comparison with literature data on the same species and data of Macrobiotus hufelandi it can be shown that eutardigrades have a roughly comparable muscular architecture, but that there are several differences in detail. Dedicated to Professor Westheide on the occasion of his 70th birthday.     

Micropropagation of <Emphasis Type="Italic">Penthorum chinense</Emphasis> through axillary bud

This study reports a protocol for successful micropropagation of Penthorum chinense using nodal explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) or kinetin (Kn). The presence of BA promoted a higher rate of shoot multiplication than Kn. Maximum multiple shoot formation was observed in 59.2% of nodal explants cultured on MS medium supplemented with 2.0 mg l⁻¹ BA after 6 wk. After subculture for 4 wk, the maximum number of shoots (6.4) was obtained on a medium with 2.0 mg l⁻¹ BA, but shoots were too short and not suitable for micropropagation. The taller shoots that regenerated in the presence of lower BA concentration (1.0 mg l⁻¹) were selected for root induction study. Most shoots (98.8%) rooted in the presence of 0.5 mg l⁻¹ indole-3-acetic acid after 3 wk, with each shoot forming an average of 10.0 roots. Plantlets were transferred to soil and successfully acclimatized.     

Influence of the<Emphasis Type="Italic">SMT2</Emphasis> knock-out on hypocotyl elongation in<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Inhibitors are very important in the study of hormone function. Brasinazole (Brz) is a specific inhibitor of brassinosteroids (BRs) biosynthesis. To expand our knowledge of the molecular mechanisms of plant steroid signaling, we performed genetic screening using medium containing Brz under dark conditions. Mutants insensitive to Brz developlonger hypocotyls than their wild type counterparts. We isolatedabz453 as a Brz insensitive mutant. TAIL-PCR and the segregation ratio of T2 plants indicated a single T-DNA insertion at the 24-Sterol C-methyltransferase (SMT2) gene in theabz453 mutant. Recapitulation for putative FCP serine phosphatase (FSP), the gene neighboringSMT2, indicated no significant phenotypes, but theSMT2 anti-sense (SMT2-AS) line developed longer hypocotyls than the wild type in medium containing Brz. Additionally, theSMT2-AS line displayed similar phenotypes to theabz453 line in soil including enhanced growth and smaller silique. Theabz453 andSMT2-AS mutants showed phenotypes similar to those of wild type in medium containing benzylaminopurine, pacrobutrazol and ACC (precursor for ethylene) under dark conditions. However, when brassinolide (BL) dose response was observed, theabz453 andSMT2-AS lines showed higher sensitivity than wild type. Theabz453/det2 andabz453/bri1-119 double mutants showed enhanced growth compared to thedet2 andbri1-119 line under both dark and light conditions. Specially, in dark conditions double mutants displayed nearly 2- and 1.5-fold longer hypocotyls thandet2 andbri1-119 plants. Brz insensitivity to theSMT2 knock-out mutant and phenotypes of double mutants indicate that not only do BRI1 and DET2 influence the BRs response, as evidenced by hypocotyl elongation, but another sterol derived signals may also be affected in mutants, suggesting that another pathway is involved in hypocotyl elongation due to SMT2.     

The incidence of mistletoe (<Emphasis Type="Italic">Viscum album</Emphasis> ssp. <Emphasis Type="Italic">abietis</Emphasis>) on silver fir (<Emphasis Type="Italic">Abies alba</Emphasis>) in Croatia

The research on incidence of mistletoe (Viscum album ssp. abietis) on silver fir (Abies alba) was carried out in natural fir stands in Croatia. In the area of Gorski Kotar 32.8 % of the examined dominant fir trees were infected with mistletoe. The mistletoe incidence was presented according to the damage degrees of silver fir from 0 (healthy trees) to 4 (dead trees), rated by the crown defoliation and needle discoloration. With the increase of incidence, mistletoe spreads more onto more vital, i.e. less damaged trees. In the Dinaric Alps 28.6% of the examined fir trees were infected, this percentage amounting to 27.1% for the mountainous regions between the Sava and Drava rivers. The site and stand parameters (exposure, elevation, site quality, forest community and crown closure) were analysed in order to establish whether there was a correlation between these parameters and mistletoe incidence. There was a negative correlation between the elevation and mistletoe incidence. Among the compartments with a closed stands there were considerably less compartments with higher mistletoe incidence than among compartments with a sparsely closed and understocked stands. The other analysed site and stand parameters individually had no significant influence on mistletoe incidence. The correlation of silver fir mortality in 2004 and mistletoe incidence in 2002/03 was analysed, and their strong correlation was established. Mistletoe could be considered as a bioindicator of silver fir decline, and probably a significant contributor to that decline. In the areas where mistletoe incidence is great it can be presumed that silver fir is significantly damaged.     

Synergistic effect between dieckol from <Emphasis Type="Italic">Ecklonia stolonifera</Emphasis> and β-lactams against methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

We have been attempting for some time to discover a compound evidencing antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The dieckol isolated from Ecklonia stolonifera has been shown to exhibit antibacterial activity against methicillin-susceptible S. aureus (MSSA) and MRSA. The minimum inhibitory concentrations (MICs) of dieckol were determined in a range of 32 to 64 μg/mL against standard MSSA and MRSA strains. Furthermore, dieckol clearly reversed the high-level ampicillin and penicillin resistance of MRSA. The MICs of ampicillin against two standard strains of MRSA were dramatically reduced from 512 to 0.5 μg/mL in combination with 1/4 MIC of dieckol (16 μg/mL). The fractional inhibitory concentration (FIC) indices of ampicillin and penicillin were measured from 0.066 to 0.266 in combination with 8 or 16 μg/mL of dieckol against all tested MRSA strains, thereby suggesting that dieckol-ampicillin or dieckol-penicillin combinations exert a synergistic effect against MRSA. The results of this study indicate that dieckol, administered in combination with β-lactams, may prove effective in the treatment of MRSA infections. Our finding may also contribute to the development of an alternative phytotherapeutic anti-MRSA agent.     

<Emphasis Type="Italic">Cardamine plumieri</Emphasis> (<Emphasis Type="Italic">Brassicaceae</Emphasis>) confirmed to the Flora of Serbia

The south-European Cardamine plumieri Vill. (sect. Pteroneurum subsect. Cryptopterum) is confirmed for the Flora of Serbia. Several populations were discovered in the central and western part of country, exclusively on the serpentine bedrocks. Morphological, chorological and ecological characteristics of C. plumieri was studied.     

Cold storage and cryopreservation of hairy root cultures of medicinal plant <Emphasis Type="Italic">Eruca sativa</Emphasis> Mill., <Emphasis Type="Italic">Astragalus membranaceus</Emphasis> and <Emphasis Type="Italic">Gentiana macrophylla</Emphasis> Pall.

To explore the possibility of an effectively long-term preservation of the germplasm of the HR lines of medicinal plant Astragalus membranaceus, Gentiana macrophylla Pall., and Eruca sativa Mill., both cold storage and cryopreservation approaches were attempted and compared. After 5-month cold storage on half strength Murashige and Skoog (1962) (1/2 MS) agar medium (AM), up to 82.9, 75.7, and 100% of the A. membranaceus, G. macrophylla and E. sativa hairy roots (HRs) recovered growth, respectively. The survival rates of A. membranaceus and G. macrophylla HRs significantly decreased, whereas that of E. sativa HR was unchanged with the addition of increased levels of exogenous abscisic acid (ABA) during cold storage. Using the encapsulation–vitrification (EV) method for cryopreservation, the G. macrophylla HRs died, whereas up to 6 and 73% of the A. membranaceus and E. sativa HRs survived, respectively. The HR lines evaluated with both methods showed no significant differences in morphology and growth rate compared with controls that were not subjected to preservation methods. These results suggest that cold storage is a more suitable alternative for the HR lines of the three studied plant species and that specificity of plant species have profound effects on the effectiveness of preservation.     

Peat-Based Inoculum of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and <Emphasis Type="Italic">Sinorhizobium fredii</Emphasis> Supplemented with Xanthan Gum

The addition of xanthan to high water retention capacity peat (HWRC) inoculants did not show differences on the survival of Bradyrhizobium japonicum E109. In low water retention capacity peats (LWRC) however, xanthan increased the survival of B.japonicum significantly. Xanthan showed the best effect at 0.1 g/l for B. japonicum, in contrast to Sinorhizobium fredii USDA205 where the concentrations evaluated (0–1.0 g/l) did not affected significantly its survival. Nevertheless, when the symbiotic performance on soybean was evaluated, the presence of 0.1 g xanthan/l increased the nodule number for both strains.     

The seasonal occurrence of endophytic fungus, <Emphasis Type="Italic">Mycosphaerella buna</Emphasis>, in Japanese beech, <Emphasis Type="Italic">Fagus crenata</Emphasis>

The seasonal occurrence of Mycosphaerella buna in leaves and contiguous organs of Fagus crenata was studied in a Japanese beech forest, Ibaraki, Japan, in 1998 and 1999. Mycosphaerella buna was not isolated from newly developed leaves in May, but it was isolated from asymptomatic leaves after June. The frequency of its occurrence gradually increased until leaffall. In contrast, M. buna was not isolated from overwintered buds, leaf petioles, or contiguous current-year twigs. The spermogonia and pseudothecia were observed in dead leaves after leaffall. The mature pseudothecia were found on dead leaves from May to July. The ascospores produced in the pseudothecia were suggested to infect newly developed leaves.Contribution no. 173, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Characterization of the nematocidal toxocyst in <Emphasis Type="Italic">Pleurotus</Emphasis> subgen. <Emphasis Type="Italic">Coremiopleurotus</Emphasis>

Toxocysts of the genus Pleurotus are blastoconidia-like ovoid structures surrounded by a liquid droplet containing a toxin that paralyzes nematodes. This study investigated toxocyst development using a strain S396 of Pleurotus cystidiosus subsp. abalonus (subgen. Coremiopleurotus). The surface of the liquid droplet was found to be an elastic envelope. When a nematode touches the toxocyst, the envelope adheres to the worm and bursts. Toxocysts are induced simultaneously with coremia formation in the absence of nematodes and developed only from aerial hyphae in which nuclear division had ceased. In the early stage of toxocyst development, liquid springs repeatedly from the tip of the sterigma-like stipe before ovoid (blastoconidium-like structure) formation. A certain substance in the liquid might polymerize to form the envelope while the ovoid simultaneously budded in the droplet. The nucleus tends to locate near the toxocyst, especially in early stage of toxocyst development. Each dikaryotic cell predominantly formed one or two toxocyst(s) while each monokaryotic cell predominantly formed one. In rare cases, a nucleus existed in the toxocyst, suggesting the possibility that the toxocyst is a vestigial blastoconidium.