Failure to decontaminate Glomus clarum NT4 spores is due to spore wall-associated bacteria

 Exposure of spores of Glomus clarum NT4 to solutions of chloramine-T (2.5–10% w/v) for 10–120 min failed to fully decontaminate all spores. Scanning electron microscopy did not show the presence of contaminants on treated spores, but transmission electron microscopy revealed bacterial cells embedded within the outer spore wall layer. Bacteria that remained protected within the spore walls were detected only when the spores were placed on appropriate media. Nutrient agar and tryptic soy agar supported relatively high levels of contaminant growth and were regarded as good media for assessing contamination, whereas the detection of contaminant growth on water agar required prolonged incubation. Contamination and germination of G. clarum NT4 spores following decontamination treatments were dependent on spore age. Generally, lower concentrations of chloramine-T and shorter incubation periods were required to reduce contamination of freshly harvested spores than of mature spores. Exposure to 10% chloramine-T for 120 min was required to reduce the levels of contamination of mature spores to ≤10%. Unfortunately, spore germination was compromised by rigorous decontamination treatments, thus the success of any decontamination procedure should be evaluated prior to its routine use. Moreover, if the interpretation of experimental results rests on the assumption of true surface sterility of VAMF spores, we suggest that the axenic condition of spores be confirmed prior to experimentation on a medium that encourages contaminant growth. Accepted: 12 July 1995     

Effect of water flow velocity on growth and morphology of cultured <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> sporophytes (Laminariales,Phaeophyceae) in Okirai Bay on the Sanriku coast,Northeast Japan

Undaria pinnatifida sporophytes, originating from the same strain, were cultured at the commercial cultivation site exposed to wave action and the uncultivated site protected from water action of Okirai Bay, Northeast Japan, from January to April 2007; simultaneously, water flow velocity, water temperature, salinity, NO₃ + NO₂, and chlorophyll a were monitored to investigate the effect of water environment on their growth and morphology. Water temperature and salinity fluctuated within the optimal range for their growth whereas water flow velocity at the cultivation site was greatly fast compared with that at the uncultivated site. Successive chlorophyll a increases synchronized with NO₃ + NO₂ decreases were observed only at the uncultivated site for over a month; indicating developments of phytoplankton blooms and their nutrient consumption under the low-flow condition. Meanwhile, blade growth rate of cultured sporophytes was higher at the cultivation site than at the uncultivated site. Their thallus size expressed by six morphological characters (blade length, stipe length, blade wet weight, stipe wet weight, blade width, and undivided blade width) at the cultivation site became large in comparison with that at the uncultivated site. Their three morphological correlations (correlations between blade length and thallus length; blade wet weight and thallus wet weight; and undivided blade width and blade width) differed between the sites. They produced a thick and flat blade at the cultivation site but formed a thin and wrinkled blade at the uncultivated site. These results show the significant impact of water flow velocity on their growth and morphology.     

A procedure for the establishment of Glomus mosseae in dual culture with Ri T-DNA-transformed carrot roots

 A stepwise procedure was investigated to determine the optimal conditions for the establishment of Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe in dual in vitro culture with Ri T-DNA-transformed roots of Daucus carota L. Glomus mosseae spores germinated best in 10 mm Tris or MES-buffered medium at pH values just above neutral. Growth of hyphae from germinated spores was much greater in the presence of Tris than MES, eg. 8 mm versus 4 mm per spore for Tris and MES, respectively, at pH 7.2. Roots exhibited a broad pH optimum for growth of 6.0–7.0 in both MES and Tris, but did not grow well above pH 7.5. In addition, purified gelling agent, gellan gum, was utilized to lower the P concentration of media. With these factors combined, mycorrhizas were successfully established in 14% of dual cultures. Accepted: 5 March 1997     

Effect of onion (Allium cepa) root exudates on the hyphal growth of Gigaspora margarita

 The effect of root exudates from onions differing in P status on spore germination and hyphal growth of arbuscular mycorrhizal fungi was investigated. Onion (Allium cepa) was grown in solution culture at different phosphorus concentrations (0, 0.1, 1.0, 8.0 and 24.0 mg P l^–1) and root exudates were collected. When spores of the arbuscular mycorrhizal fungus, Gigaspora margarita were incubated with these root exudates, spore germination was only slightly affected but hyphal growth was greatly affected, particularly with exudates from P-deficient plants. This suggests that the P nutrition of host plants influences the composition of root exudates and thereby the hyphal growth of arbuscular mycorrhizal fungi. Accepted: 25 June 1995     

Indigenous populations of arbuscular mycorrhizal fungi and soil aggregate stability are major determinants of leek (Allium porrum L.) response to inoculation with Glomus intraradices Schenck & Smith or Glomus versiforme (Karsten) Berch

 Knowledge of physical, chemical and biological soil characteristics influencing plant response to inoculation with arbuscular mycorrhizal (AM) fungi would help to distinguish soils where inoculation could be profitable. The relationship between leek (Allium porrum L.) response to mycorrhizal inoculation with Glomus intraradices Schenck & Smith or G. versiforme (Karsten) Berch and soil texture, bulk density, particle density, porosity, pH, organic matter content, available P, K, Ca, Mg, Fe, Zn, Cu, and Mn, soil structure, soil mycorrhizal potential (SM), preceding crop mycorrhizal potential, composition of indigenous mycorrhizal fungal communities, and the abundance of spores of different species, was studied in 81 agricultural soils using Principal Component Analysis and regression analysis. The nature of the indigenous AM fungi population was an important determinant of leek response to inoculation (RTI). In soils with more than 200 μg available P g^–1, SM potential accounted for over 27% of RTI with G. intraradices and G. versiforme, RTI being high in soils with low SM potential. In low P soils, however, a positive relation between the abundance of water stable soil aggregates in the 0.5–2 mm diameter range and RTI was most important. Low soil Zn and high porosity, abundant total mycorrhizal spore as well as scarcity of spores of G. aggregatum and of the group G. etunicatum-rubiforme were also associated to high RTI. The influence of water stable aggregation of soil on RTI was modulated by soil P levels. Abundance of soil aggregates was positively related to RTI at low soil P levels, but negatively related to RTI at high P levels. Different relationships were found between soil variables and spore abundance of different AM fungi species. Some AM species appear to have as yet undefined similarities or complementarities at the biological or ecological levels. Accepted: 23 July 1997     

Sporulation and regeneration efficiency of phosphobacteria (<Emphasis Type="Italic">Bacillus megaterium</Emphasis> var <Emphasis Type="Italic">phosphaticum</Emphasis>)

Sporulation in Bacillus megaterium var phosphaticum (PB — 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB — 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10⁸ cfu ml⁻¹) in nutrient broth containing 5 per cent inoculum level.     

Effect of root exudate fractions from P-deficient and P-sufficient onion plants on root colonisation by the arbuscular mycorrhizal fungus Gigaspora margarita

 The effect of root exudates from P-deficient onion on root colonisation by an arbuscular mycorrhizal fungus was examined. Onions (Allium cepa L.) were grown in solution culture at phosphorus concentrations of 0 (P0) and 2 (P2) mg P l^–1. Root exudates were collected and fractionated with Amberlite XAD-4 resin to give EtOH and water soluble fractions. Onions inoculated with the arbuscular mycorrhizal fungus Gigaspora margarita Becker & Hall were grown with or without (control) root exudates and exudate fractions in a growth chamber. After 24 days, arbuscular mycorrhiza levels and appressoria formation had increased in plants treated with P0-root exudate or the P0-EtOH fraction when compared to corresponding P2 treatments or control plants. P0 and P2 water-soluble fractions did not significantly affect either aspect of fungal development. These results suggest that hydrophobic compounds found in root exudates from P-deficient onion increase appressorium formation and, therefore, enhance mycorrhiza development. Accepted: 2 June 1998     

The significance of thallus size for the water economy of the cyanobacterial old-forest lichen Degelia plumbea

Rosette-formed, circular thalli of Degelia plumbea were studied in the laboratory. Regardless of thallus size, the optimal quantum yield of photosystem II (F _V/F _M) remained at a high, constant level during a drying cycle starting with fully hydrated thalli until the thallus water content reached about 200%. Net photosynthesis reached a maximum level at this hydration level. Thereafter, both F _V/F _M and net photosynthesis fell rapidly to zero at a water content of somewhat less than 100%. There was a highly significant, positive relationship between thallus size and the water-holding capacity, as well as a strong, negative correlation between size and water loss per thallus area. Consequently, an increase in thallus size from 1 to 36 cm² lead to a tenfold prolongation of the photosynthetically active period during a drying cycle at a low radiation regime. The improved water-holding capacity in larger thalli is mainly a result of a thicker hypothallus. The fast desiccation of small thalli suggests that the regeneration of D. plumbea could be severely hampered by nearby logging that raises the evaporative demand by increasing radiation loads and wind exposure at remaining lichen sites. Received: 12 December 1997 / Accepted: 20 March 1998     

Infectivity of monoaxenic subcultures of the arbuscular mycorrhizal fungus Glomus versiforme associated with Ri-T-DNA-transformed carrot root

  Glomus versiforme was associated in vitro with Ri-T-DNA-transformed carrot root and after 4 months of cultivation, numerous axenic arbuscular mycorrhizal (AM) propagules were obtained. Three successive generations of spores and mycorrhizal root pieces were obtained by reassociating a 4-month-old root piece with a new carrot root. A biological test was conducted to assess the infectivity of the three generations of inoculum. Both for spores and mycorrhizal root pieces, a significant decrease in infectivity was observed among the successive generations. Mycorrhizal root pieces however, exhibited, higher infectivity than spores. These results show the possibility of maintaining AM fungi cultures in axenic conditions for a long time and raise questions about the loss of infectivity among the generations. The necessity of maintaining the same strains in vitro and in vivo in two separate collections is discussed. Received: 15 January 1996 / Received revision: 28 May 1996 / Accepted: 16 June 1996     

Effects of arbuscular mycorrhizal fungi on seedling growth and development of two wetland plants, Bidens frondosa L., and Eclipta prostrata (L.) L., grown under three levels of water availability

To identify the importance of arbuscular mycorrhizal fungi (AMF) colonizing wetland seedlings following flooding, we assessed the effects of AMF on seedling establishment of two pioneer species, Bidens frondosa and Eclipta prostrata grown under three levels of water availability and ask: (1) Do inoculated seedlings differ in growth and development from non-inoculated plants? (2) Are the effects of inoculation and degree of colonization dependent on water availability? (3) Do plant responses to inoculation differ between two closely related species? Inoculation had no detectable effects on shoot height, or plant biomass but did affect biomass partitioning and root morphology in a species-specific manner. Shoot/root ratios were significantly lower in non-inoculated E. prostrata plants compared with inoculated plants (0.381 ± 0.066 vs. 0.683 ± 0.132). Root length and surface area were greater in non-inoculated E. prostrata (259.55 ± 33.78 cm vs. 194.64 ± 27.45 cm and 54.91 ± 7.628 cm² vs. 46.26 ± 6.8 cm², respectively). Inoculation had no detectable effect on B. frondosa root length, volume, or surface area. AMF associations formed at all levels of water availability. Hyphal, arbuscular, and vesicular colonization levels were greater in dry compared with intermediate and flooded treatments. Measures of mycorrhizal responsiveness were significantly depressed in E. prostrata compared with B. frondosa for total fresh weight (−0.3 ± 0.18 g vs. 0.06 ± 0.06 g), root length (−0.78 ± 0.28 cm vs.−0.11 ± 0.07 cm), root volume (−0.49 ± 0.22 cm³ vs. 0.06 ± 0.07 cm³), and surface area (−0.59 ± 0.23 cm² vs.−0.03 ± 0.08 cm²). Given the disparity in species response to AMF inoculation, events that alter AMF prevalence in wetlands could significantly alter plant community structure by directly affecting seedling growth and development.     

ThePorphyra species of Helgoland (Bangiales,Rhodophyta)

This revision of sevenPorphyra species of Helgoland was based on a study of the structure of their fertile thalli and the behaviour of their spores. Regarding the reproductive organization the species may be arranged in two groups.P. leucosticta andP. purpureo-violacea are obligate monoecious species. Asexual thalli have never been observed in the field. The other five species are generally dioecious. Isomorphic sexual thalli and asexually propagating ones are mixed in uniform populations. Carpospores originating from sexual fusion develop into the diploid Conchocelis phase. Sporangia of asexual plants, though homologous in formation, produce spores of different kinds: aplanospores that give rise to the vegetative thallus directly (inP. umbilicalis, P. insolita n. sp. andP. ochotensis) and spores that develop into haploid Conchocelis (inP. laciniata and inP. linearis). P. laciniata — formerly considered synonymous withP. purpurea — is an independent species. “In an investigation into the life-history of an alga the aim should be to obtain a series of observations of the development of the thallus from the germinating spore and then of the development of the reproductive organs on the mature thallus. In cases where both sexual and asexual spores are formed the germination of both types of spore should be followed, ...” (Drew, 1954, p. 184)     

Effects of arbuscular mycorrhizal fungi on tree growth, leaf water potential, and levels of 1-aminocyclopropane-1-carboxylic acid and ethylene in the roots of papaya under water-stress conditions

 Seedlings of papaya (Carica papaya L. var. Solo) were transplanted to pots with or without an arbuscular mycorrhizal (AM) fungus (Gigaspora margarita Becker and Hall). After 3 months, half the plants were subjected to water stress by withdrawing irrigation. The leaf water potential (LWP) was measured during 20 days of water-stress treatment and then the plants were harvested. Root ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC) concentrations were measured and plant fresh weight determined. The LWP decreased during the water-stress treatment and this decrease was more severe in the non-AM plants. Plant fresh weight was higher for AM than non-AM plants under both conditions. Under well-irrigated conditions, the ethylene concentration in the roots was increased by the presence of AM, although there was no significant difference between AM and non-AM roots in ACC levels. ACC increased in both AM and non-AM roots under water-stress conditions. The water-stress treatment resulted in a marked increase in ethylene concentration in non-AM roots but the concentration in AM roots was slightly lower than under normal conditions. Accepted: 7 July 2000     

UVB effects on early developmental stages of commercially important macroalgae in southern Chile

High levels of ultraviolet-B radiation (UVB) could represent a danger to seaweeds by affecting their physiological processes and development. The aim of this work was to study the effects of UVB radiation on early developmental stages of commercially and ecologically important marine algal species in southern Chile, considering spores survival and embryos growth. Spores of Mazzaella laminarioides, Gigartina skottsbergii, Sarcothalia crispata and embryos of G. skottsbergii and Macrocystis pyrifera were submitted to treatments of a) photosynthetically active radiation (PAR: Control), b) PAR+UVA (PA) and c) PAR+UVA+UVB (PAB). UV radiation did not affect spore survival of M. laminarioides S. crispata and G. skottsbergii (P = 0.55, P = 0.6 & P = 0.25 respectively), but did provoke differences in the growth rate of G. skottsbergii embryos (P = 0.00). Differences in survival and growth of M. pyrifera embryos were also observed (P = 0.001 & P = 0.007, respectively). Differences in growth of M. pyrifera embryos were observed only in the first five days, whereas changes in survival persisted until the end of the experiment. Additionally, UVB provoked morphological alteration in M. pyrifera embryos, as evidenced by progressive curling. These results suggest that the initial stages of the subtidal algae species G. skottsbergii and M. pyrifera cultivated in laboratory conditions were sensitive to UVA and UVB radiation, and their recruitment and development could be affected as well in natural conditions found in southern South America, where the ozone layer has thinned more than in other parts of the planet.     

Continuous marennin production by agar-entrapped Haslea ostrearia using a tubular photobioreactor with internal illumination

The marine diatom Haslea ostrearia was immobilized in a tubular agar gel layer introduced into a photobioreactor of original design with internal illumination for the continuous synthesis of marennin, a blue-green pigment of biotechnological interest. Marennin was produced for a long-term period (27–43 days) and the volumetric productivity was maximum (18.7 mg day⁻¹ l⁻¹ gel) at the highest dilution rate (0.25 day⁻¹) and lowest agar layer thickness (3 mm). Heterogeneous cell distribution in the agar layer revealed diffusional limitation of light and nutrients. However, the 3 mm gel thickness led to a more homogeneous cell distribution during incubation and to an increase of the whole biomass in the agar gel layer. Received: 22 October 1999 / Received revision: 14 February 2000 / Accepted: 18 February 2000     

Short communication: Influence of inoculum preparation on citric acid production by Aspergillus niger

The type of sporulation medium and time of incubation had an effect on spore viability and citric acid production by mycelia grown from Aspergillus niger spores. Shu & Johnson agar (SJA) and potato dextrose agar gave higher citric acid titres than malt-extract agar. SJA also gave better germinability than the other media. Viability increased with time of incubation, but higher production of citric acid was achieved with spores incubated for less than 7 days.     

Preparation of superior quality products from two Indian agarophytes

The effect of alkali pre-treatments on the properties of agar obtained from Gelidiella acerosa and Gelidium pusillum growing along the West and South coast of India was studied in order to evaluate its potential for industrial use. Agar samples were extracted by hot water extraction and termed native agar, and alkali pre-treatment was carried out with various NaOH concentrations e.g. 4%, 6%, 8%, 10% and 15%, to optimise the alkali concentration for preparation of alkali-treated agar. Gelidiella acerosa and Gelidium pusillum produced superior quality agar polymers having greater gel strengths 2,000 ± 50 and 1,400 ± 50 g cm⁻² and lower sulphate contents ≤0.30% and ≤0.50%, respectively, as opposed the low gel strength and high sulphate content agars reported in the literature. Furthermore, the physicochemical properties of agars were identical with the commercially available agars. The results of this study revealed that Indian agarophytes may be used for production of superior quality agar and agaroses, which may be used for food and biological applications.     

Clonal propagation of mesquite tree (<Emphasis Type="Italic">Prosopis laevigata</Emphasis> Humb. &; Bonpl. ex Willd. M.C. Johnston). I. Via cotyledonary nodes

Plantlet regeneration in Prosopis laevigata (Humb. & Bonpl. ex Willd.) Johnston (Fabaceae), a multipurpose tree, has been achieved from cotyledonary nodes excised from in vitro grown seedlings. The explants were cultured on MS media containing different concentrations of N-6 benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-d) and a mixture of organic components. The highest number (3.37 + 0.51) of multiple shoots was observed in MS media containing 2,4-d (9.05 μM) + BA (6.62 μM). The regenerated shoots were then transferred onto half-strength MS medium containing a plant growth regulator that was either: indole-3-butyric acid, 1-naphthaleneacetic, indole-3-acetic acid, or 2,4-d as well as phytagel or vermiculite for adventitious root initiation. Best rooting efficiency of 44.0% was obtained when NAA (16.11 μM) and vermiculite were used. After rooting, the cloned plantlets were successfully hardened to ex vitro conditions. This work may help to reduce the devastation caused by the overexploitation of this species.     

Endogenous hormone levels in habituated nucellar Citrus callus during the initial stages of regeneration

 Embryogenic nucellar callus cultures of different Citrus species and cultivars growing in hormone-free medium were transferred to medium containing either sucrose or glycerol as the only carbohydrate source. Glycerol has been reported to induce further development of Citrus somatic embryos, while in the presence of sucrose they continue to proliferate in an 'undifferentiated' manner. The endogenous hormone levels of the cultures were evaluated after 2 and 5  days to characterise the initial steps of embryo development. In most cases, differences among treatments were observed only after 5 days of culture. Higher cytokinin levels were found in most of the cultures transferred to the glycerol-containing medium. The effect of ageing sweet orange cultures on their endogenous hormone levels was determined by leaving them in the original culture medium without subculturing for 60 days. While no changes were observed in the free indoleacetic acid and gibberellin contents, lower levels of abscisic acid and cytokinins were found in the aged cultures than in those transferred at the normal interval, every 30 days. The endogenous hormone contents of Citrus callus of different genotypes were compared. Significant differences were observed in the levels of all hormones evaluated, even when the in vitro ontogeny of the different genotypes was very similar. Received: 10 February 2000 / Revision received: 25 August 2000 / Accepted: 29 August 2000     

The A mating type and blue light regulate all known differentiation processes in the basidiomycete Coprinus cinereus

Monokaryons of Coprinus cinereus constitutively form small spores (oidia) in the aerial mycelium. Some strains also produce large, inflated single cells (chlamydospores) at the agar/air interface, and hyphal aggregates (hyphal knots) that can develop into sclerotia. Monokaryons show various reactions upon transformation with heterologous A mating type genes. Production of oidia in such A-activated transformants is repressed in the dark and induced by blue light. Five of six monokaryons tested following transformation with A genes showed induced production of hyphal knots and sclerotia in the dark, and at least three strains showed enhanced chlamydospore production in the dark. Continuous incubation under blue light inhibited formation of hyphal knots, sclerotia and chlamydospores in both competent monokaryons and in A-activated transformants. On artificial medium and on a 12 h light/12 h dark regime, A-activated transformants of one distinct monokaryon (218) formed fruit-body primordia that were arrested in development before karyogamy. Our studies show that A mating type genes control all major differentiation processes in Coprinus, but whether developmental processes can proceed depends on the genetic background of the strain. Received: 11 May 1998 / Accepted: 15 July 1998     

Heat resistance of Paecilomyces variotii in sauce and juice

It has been demonstrated that some anamorphic fungi ( Paecilomyces variotii, Fusarium sp) could cause spoilage of food products after pasteurisation. Four food-borne and one clinical isolate of P. variotii were cultivated on one solid medium and three liquid media. Their survival after heating at 80–100˚C for 0.25–15 min in sterile distilled water and curry sauce or fruit juice was investigated. Heat resistance was determined by the thermal death method in a thermostatically-controlled oil bath. The most resistant spores of P. variotii from curry sauce cultivated on malt extract agar survived 100˚C for 0.5 min in sauce; cultivated in curry sauce survived 100˚C for 15 min in water and cultivated in malt broth survived 100˚C for 5 min in water and sauce. The most resistant spores of P. variotii from juice cultivated on malt extract agar were able to survive 100˚C for 15 min in water; cultivated in juice survived 100˚C for 0.5 min in juice and suspensions from cultivation in malt broth survived 100˚C for 1.5 min in juice. Spores of the clinical strain of P. variotiifrom malt extract agar survived 95˚C for 0.33 min in water, and orange juice cultures survived 96˚C for 10 min in orange juice. It was thus found that P. variotii strains cultivated in food were better adapted to heat stress, suggesting that fungal biomass suspensions were able to survive the higher temperatures for longer time intervals than spore suspensions. Journal of Industrial Microbiology & Biotechnology (2000) 24, 227–230. Received 02 June 1999/ Accepted in revised form 05 December 1999