Atkinsiella awabi sp. nov. isolated from stocked abalone,Haliotis sieboldii

A fungal disease in the abalone,Haliotis sieboldii, stocked in Yamaguchi Prefecture, Japan, showed external signs of infection of tubercle-like swelling on the mantle and melanized lesions on the peduncle. The fungus responsible was isolated by inoculating materials taken from the lesions onto PYGS agar with streptomycin sulphate and ampicillin, and incubation at 20°C. For morphological observation and spore formation study, the fungus was transferred respectively into PYGS broth and sterilized artificial seawater and incubated at 20°C. Resulting, hyphae were stout, irregular, branched, 16–140µm diam, sporadically consisting of dense cytoplasmic swollen hyphae. Sporangia were formed through the formation of septa and lateral or terminal discharge tubes which were wavy or coiled. Zoospores were pyriform, biflagellate and diplanetic. The encysted spore generally developed a hairlike filament with globular enlarged tip in PYGS broth. Direct germination without filament formation also occurred occasionally. This fungus was identified as belonging to the genusAtkinsiella, and was designatedAtkinsiella awabi sp. nov. The fungus was exclusively a marine fungus and grew best in shrimp extract medium at 20°C. Five chemicals were tested for their effects against fungal zoospores.     

Biological control of alligatorweed (Alternanthera philoxeroides) with a Fusarium sp.

Alligatorweed (Alternanthera philoxeroidesG.) has become a serious weed in different crops in China. A fungal pathogen was found in Chongqing and Sichuan Provinces and was identified as a species in the Fusarium genus. The fungus produced macroconidia and chlamydospores abundantly on potato sucrose agar (PSA) plates. The bestconidial production and germination and colonygrowth of Fusarium sp. were at 23–31°C and pH 6.7–7.0. Light period and flooding did not affect fungal growth and conidium formation. The herbicides, glyphosate and paraquat, inhibited the fungal development in vitro. The fungus did not affect seed germination and seedling growth of paddy rice, wheat, maize, oilseed rape and broad bean inlaboratory or greenhouse trials. Inoculum density and wetness duration influenced the efficiency of Fusarium sp. to control alligatorweed; a concentration of 1.0 × 10⁵ spores^–1 ml and 12 h of high humidity duration after inoculation produced goodinfections on the weed at 23°C in the laboratory. When the fungus was applied to alligatorweed grown in greenhouse and in the field, good biocontrol efficiency was obtained: the plants started to wilt after four to five (greenhouse) or six days (field), and were killed 9–10 (greenhouse) or 13–14 (field) days after spraying the fungal inoculum. This was similar to the control efficiency resulting from glyphosate treatment. Therefore, this Fusarium sp. appeared to be a good candidatefor further studies and a promising biocontrol agent to manage alligatorweed in some terrestrial and aquatic crops.     

Stimulation of artemisinin production in Artemisia annua hairy roots by the elicitor from the endophytic Colletotrichum sp.

Artemisinin content in hairy roots of Artemisia annua was increased from 0.8 mg g^–1 dry wt to 1 mg g^–1 dry wt by using elicitor treatment of mycelial extracts from the endophytic fungus Colletotrichum sp. The increase of artemisinin was dependent on the growth stage of hairy roots as well as on the dose of the elicitor applied. When hairy roots of 23-day-old cultures (later growth phase) were exposed to the elicitor at 0.4 mg total sugar ml^–1 for 4 days, the maximum production of artemisinin reached to 13 mg l^–1, a 44% increase over the control. This is the first report on the stimulation of artemisinin production in hairy roots by the elicitor from an endophytic fungus of A. annua.     

The effects of the physical characteristics of the culture medium on the development of red seaweeds in tissue culture

Explants of Gelidium versicolor, Grateloupia doryphora and Laurencia sp. were cultivated in Provasoli enriched seawater culture medium (PES) adjusted to several osmolalities (0.5, 0.7, 1.0 and 1.5 Os kg^–1) and solidities (agar concentration = 3, 8 and 15 g L^–1). Osmolality was adjusted by dilution of seawater with distilled water (50, 70 and 100% seawater) and by NaCl addition. Explants of Laurencia sp. and Grateloupia doryphora showed bud regeneration and callus formation. Explants of Gelidium versicolor only showed bud regeneration. Osmolalities of 0.5 and 1.05 Os kg^–1. inhibited or drastically reduced bud regeneration and callus formation. The highest callus formation and bud regeneration were observed at 0.7 to 1.0 Os kg^–1. An increase in the agar concentration of the culture medium was positively correlated with callus formation and negatively correlated with bud regeneration. An increase in the percentage of seawater increased the solidity of the culture medium and was positively correlated with callus formation. Glycerol was an effective carbon source for the vegetative propagation of axenic explants of Grateloupia doryphora, promoting growth and bud regeneration. An increase in glycerol concentration in the culture medium increased its osmolality, inhibiting the growth of the explants and their morphogenetic development.     

Lagenidium myophilum infection in the coonstripe shrimp,Pandalus hypsinotus

A fungal infection occurred in juvenile coonstripe shrimps,Pandalus hypsinotus, cultured at Hokkaido Institute of Mariculture, Hokkaido, Japan. The fungus was identified asLagenidium myophilum, the same fungus that had previously been isolated from the abdominal muscle of adult northern shrimps,Pandalus borealis, and larvae of the coonstripe shrimp. Histopathologically, numerous nonseptate hyphae were observed in the lesions, and melanized hemocytes were present within the blackened areas. The optimum temperature for growth of the present strain was 25–30°C, and the optimum NaCl concentration for growth was 0.5–1.0%. Its biological characteristics were compared with those ofLagenidium myophilum isolated from diseased larval coonstripe shrimp and adult northern shrimp. The fungus was pathogenic toward shrimps of the genusPandalus, which live in deep sea areas. The fungus could infect shrimps at various stages, from larva to adult.     

In vitro zygotic embryo culture of an endangered forest tree Givotia rottleriformis and factors affecting its germination and seedling growth

Summary This study reports a protocol for germination of Givotia rottleriformis (var. Tel. Thella Poniki) using zygotic embryo culture. A 100% germination was obtained by culturing the embryos on Murashige and Skoog medium containing 30 gl^−1 sucrose. A sucrose concentration lower or higher than 30 gl^−1 resulted in lower germination or promoted callus formation. The seedling growth was promoted by the addition of 100 mgl^−1 tyrosine in the medium. Seedlings germinated in the presence of 0.2–0.4 mgl^−1 α-naphthaleneacetic acid and 0.3–0.5 mgl^−1 indole-3-butyric acid were abnormal, showing a slender stem with slender roots or forming callus with stout roots. Germination also affected embryo orientation in culture; placing embryos upright on the medium was most beneficial for germination. The in vitro-germinated seedlings were acclimatized in soil under shady conditions with a survival rate of 60–70%. These plants were phenotypically normal, healthy, and similar to donor plants. This protocol will be useful for overcoming seed dormancy and for rapid multiplication and conservation of G. rottleriformis using zygotic embryo culture.     

Studies on the production of useful chemicals,especially fatty acids in the marine diatom Nitzschia conspicua Grunow

A local marine diatom, Nitzschia conspicua Grunow, was cultured in enriched synthetic seawater using flasks (agitated by magnetic stirring) and a 1.2 l fermenter. Lipids, fatty acids, proteins, carbohydrates and ash of the flask cultures were determined at various stages of growth (day 3, 5, 7, 10, 13, 15 and 17). The fermenter culture was harvested during the stationary phase for similar chemical analyses. N. conspicua attained a higher biomass concentration during the stationary phase when cultured in the fermenter (188 mg dry weight l^–1) than in flasks (140–151 mg dry weight l^–1). However, both systems showed similar specific growth rates based on chlorophyll-a concentration. Appreciable amounts of the essential fatty acids 20:4 (0.6–4.7% total fatty acids) and 20:5 (1.9–4.7% total fatty acids) are present in this diatom. Maximal amounts of these fatty acids were produced after 7 days' growth (i.e. 2 days after the end of the exponential phase). Lipids, fatty acids, proteins, carbohydrates and ash varied with culture age in N. conspicua.author for correspondence     

Identity and frequency of occurrence ofTrichoderma spp. in roots of wheat and rye-grass in Western Australia and their effect on root rot caused byGaeumannomyces graminis var.tritici

Trichoderma hamatum, T. harzianum andT. koningii were isolated from wheat and rye-grass roots from a field in Western Australia. Frequency of occurrence ofTrichoderma spp. was higher on roots subjected to washing only, for both wheat and rye-grass than the roots which were surface-sterilized with 0.6% or 1.25% NaOCl.Trichoderma spp. were recovered at a higher frequency on PDA amended with lactic acid (pH 4.5) than on PDA alone (pH 5.6) or PDA with streptomycin. In general,Trichoderma spp. were isolated at a higher frequency from roots of wheat than that of rye-grass.T. hamatum occurred at a higher frequency in rye-grass roots than in wheat, whereasT. harzianum was more common in roots of wheat than in rye-grass, especially in seedling and milky ripe stages.T. koningii was recovered at a higher frequency from roots at seedling stage of rye-grass than wheat, the reverse being true at tillering stage.T. koningii was not recovered from roots of either host in any sampling when they were surface sterilized with 1.25% NaOCl.The take-all fungus was isolated from wheat and rye-grass roots more frequently at tillering and stem extension stages than others. It was severely pathogenic to both hosts in sterilized and non-sterilized soil.Addition of lactic acid, HCl or streptomycin to PDA did not affect the growth of theTrichoderma spp. tested, but the growth was slower on Martin's medium than on other media. In generalT. harzianum andT. koningii grow faster thanT. hamatum. The growth of the three species were not different at 20 and 25°C, but at 15°c growing of all species was significantly reduced.Incorporation of lactic acid into PDA prevented the bacterial growth in all treatments. Streptomycin too reduced but to a lesser degree than lactic acid. Surface sterilization with NaOCl decreased the recovery of both bacteria and fungi. T. hamatum andT. koningii reduced the mortality of wheat and rye-grass plants inoculated with the take-all fungus in sterilized and non-sterilized soil, whereT. harzianum did not protect wheat or rye-grass from infection by the take-all fungus.     

Morphogenetic effect of glycerol on tissue cultures of the red seaweed Grateloupia doryphora

Explants of Grateloupia doryphora were cultivated in Provasoli Enriched Seawater culture medium (PES) supplemented with glycerol (0.1, 0.3, 0.5 or 0.8 mol 1^–1) or carbohydrates (0.1 or 0.3 mol 1^–1 mannose, glucose and galactose) and agar (3, 8, 15 g 1^–1 ). The osmolality of the medium was adjusted by dilution of the seawater (70 or 100%, v/v). The increase in fresh weight of explants cultivated in liquid medium with glycerol (0.3 mol 1^–1) and without glycerol was compared. All experiments were carried out in the light, except for one assay in which the explants were cultivated in the dark. Glycerol was an effective carbon source for the vegetative propagation of G. doryphora in solid and liquid media. Mannose, glucose and galactose all had no effect on growth or morphogenesis of the explants. In solid media the main effect of glycerol was as a morphogenetic inductor, with PES70 (70% seawater) + 0.1 or 0.3 mol 1^–1 glycerol + 3 or 8 g 1^–1 agar the best formulation. An increase in the concentration of agar in glycerol-containing medium reduced the morphogenetic capacity of the explants, which developed into compact cell masses. The effects of glycerol were observed only in explants cultivated under light.     

In vitro evaluation of combination of Trichoderma harzianum and chitosan for the control of sapstain fungi

In vitro assays were undertaken to evaluate the control of two sapstain fungi, Leptographium procerum and Sphaeropsis sapinea by a combination of chitosan or chitosan oligomer and an albino strain of Trichoderma harzianum. Spore germination and hyphal growth of the test fungi were assessed on media amended with chitosan or chitosan oligomer with and without T. harzianum using either simultaneous inoculation with test fungus or inoculation 1, 2, or 3 days after pre-infection with test fungus.There was no mycelial growth of the test fungi regardless of chitosan concentrations used when either L. procerum or S. sapinea was simultaneously inoculated with T. harzianum. However, the dose–response of chitosan or chitosan oligomer on the test fungi was apparent when T. harzianum was not simultaneously inoculated with test fungus but introduced later. There was a greater growth reduction at higher concentrations (0.075–0.1% v/v) of chitosan, and overall chitosan oligomer was more effective than chitosan aqueous solution.Chitosan alone was able to restrict or delay the germination of spores but the combination of chitosan and T. harzianum inhibited spore germination and hence colony formation of test fungi regardless of time delay.     

Atkinsiella parasitica sp. nov. isolated from a rotifer,Brachionus plicatilis

A new species ofAtkinsiella (Lagenidiales, Oomycetes),Atkinsiella parasitica is described and illustrated. It was isolated from the eggs and bodies of a rotifer,Brachionus plicatilis, with fungal infection in 1992. The rotifer was reared in a hatchery as the first food supply for seed production of crustaceans and fishes. The fungus is characterized by producing monoplanetic, lateral biflagellate zoospores, and infrequently branched discharge tubes. Optimum temperature for the fungus was 25°C. The fungus was considered an obligate marine fungus, because its growth was observed only on PYGS medium including seawater.     

Lagenidium infection in eggs and larvae of mangrove crab (Scylla serrata) produced in Indonesia

A fungal infection occurred in the eggs and larvae of mangrove crab (Scylla serrata) in seed production in Bali, Indonesia. The causative fungus was classified as a member of the genusLagenidium (Oomycetes, Lagenidiales). After comparison of its biological and physiological characteristics with those ofL. callinectes ATCC 24973, a known parasite of various crustaceans, was concluded that the isolate is a new species ofLagenidium, L. thermophilum, because of its rapid and thermotolerant growth and unique discharge process. Fungal growth was observed on PYG agar containing 0–5.0% (w/v) NaCl and 0–2.5% (w/v) KCI. Similar pathogenicity toward the zoeae of swimming crab (Portunus trituberculatus) was demonstrated.     

Development and reproduction of Stratiolaelaps scimitus (Acari: Laelapidae) with fungus gnat larvae (Diptera: Sciaridae), potworms (Oligochaeta: Enchytraeidae) or Sancassania aff. sphaerogaster (Acari: Acaridae) as the sole food source

Stratiolaelaps ( = Hypoaspis) miles (Berlese) (Acari: Mesostigmata: Laelapidae) is a polyphagous soil-dwelling predatory mite that is widely marketed for use in greenhouse production systems to manage populations of dark-winged fungus gnats, Bradysia spp. (Diptera: Sciaridae) and for supplemental control of thrips. The suggestion by Walter and Campbell (2003, Biol. Control 26: 253–269) that North American commercial cultures of S. miles may actually be S. scimitus was confirmed. The development and reproduction at 21–23 °C of S. scimitus provided ad libidum with one of three different prey – the fungus gnat Bradysia aff. coprophila (Lintner), potworms (Enchytraeidae), or Sancassania aff. sphaerogaster (Zachvatkin, 1937) (Acari: Astigmata: Acaridae) – were compared. Developmental duration of the egg and non-feeding larval stages were 2.47 and 1.11 days, respectively; mortalities were 8.3 and 5.5%. Stratiolaelaps scimitus failed to develop beyond the protonymphal stage when provided with S. aff. sphaerogaster alone, although some feeding was observed. Development and reproduction of S. scimitus was successful on both fungus gnat larvae and enchytraeids, with no influence of prey on protonymphal duration (4.70 days) and mortality (8.3%), or on deutonymphal duration (4.61 days) and mortality (6.1%). Adult female S. scimitus feeding on potworms, compared to those feeding on fungus gnat larvae, had a significantly shorter pre-oviposition period (2.69 vs. 4.59 days). However, diet did not influence other adult female developmental or reproductive characteristics (oviposition period, 18.6 days; post-oviposition period, 6.2 days; total adult longevity, 27.3 days; total number of eggs, 26.5). S. scimitus reared on potworms tended (p = 0.06) to have a higher intrinsic rate of increase, a higher finite rate of increase and a shorter doubling time (r _m = 0.142 day⁻¹, λ = 1.153, Dt = 4.85 days) than those reared on fungus gnat larvae (r _m = 0.105 day⁻¹, λ = 1.110, Dt = 6.58 days), but differences in net reproductive rate (R ₀) and generation time (G) were not significant.     

Field trials against Hoplia philanthus (Coleoptera: Scarabaeidae) with a combination of an entomopathogenic nematode and the fungus Metarhizium anisopliae CLO 53

The white grub, Hoplia philanthus Füessly (Coleoptera: Scarabaeidae), is a major pest of turf and ornamental plants in Belgium. Previously, the combination of lethal concentration of the entomopathogenic nematodes Heterorhabditis megidis or Steinernema glaseri with the entomopathogenic fungus Metarhizium anisopliae (strain CLO 53) caused additive or synergistic mortality to third-instar H. philanthus in the laboratory and greenhouse. In this present study, we examined this interaction under field conditions and compared a combination of a commercial formulation of Heterorhabditis bacteriophora (Nema-green^®) and M. anisopliae. Controls were M. anisopliae, chlorpyrifos (Dursban 5 Granules) and H. bacteriophora. Field applications (surface or subsurface) were made against a mixed population of second/third-instar H. philanthus at a sport field and lawn infested in the province of West-Flanders. In both trials, the combination of M. anisopliae with H. bacteriophora at 5 × 10¹² conidia/ha +2.5 × 10⁹ infective juveniles/ha resulted in additive or synergistic effects, causing more than 95% grub mortality when the nematodes was applied 4 weeks after the application of fungus. However, application of nematode, chlorpyrifos or fungus alone provided 39–66%, 42–60% (surface) and 33–76%, 82–100% or 37–65%, (subsurface) control of H. philanthus. We concluded that the pathogen combinations we tested are compatible elements of integrated pest management and are likely to improve control of H. philanthus larvae and perhaps other insect pests beyond what is expected from single application of the pathogen.     

Environmental effects on growth and biochemical composition ofNitzschia inconspicua Grunow

The effects of nitrate and silicate levels, and carbon source on growth, biochemical composition and fatty acid composition ofNitzschia inconspicua were investigated using batch cultures. Within the range of silicate levels supplied (8.8–176 M), no marked variations in growth trend, biochemical composition or fatty acid composition were shown. Biomass at stationary phase, ranging from 64–66 mg ash-free dry weight (AFDW) L^–1, and specific growth rate () based on chlorophylla (0.41–0.50 d^–1) of the cultures grown within 0.3–3.0 mM NaNO₃ were not significantly different. Cultures supplemented with glucose (0.1 % w/v), acetate (0.1 % w/v) or 5% CO₂ attained higher biomass (85, 85, 97 mg AFDW L^–1) than the control which was grown in synthetic seawater and agitated by magnetic stirring. Cells grown at <3.0 mM NaNO₃ contained higher carbohydrate contents (14.8–21.5% AFDW) than those grown at 3.0 mM (4.0% AFDW). Lipid content increased at the expense of proteins in cells aerated with 5% CO₂. The dominant fatty acids, 16:0 and 16:1, ranged from 35.7–45.0% and 36.4–45.4% total fatty acids (TFA), respectively, while the relative proportions of 20:4 (n-6) and 20:5 (n-3) ranged from 1.7–5.4% and 3.4–5.9% TFA respectively. Cultures aerated with 5% CO₂ attained the highest biomass (97 mg AFDW L^–1) and yield of 20:5 (n-3) (0.34 mg L^–1).     

Clonal propagation of Eucheuma denticulatum and Kappaphycus alvarezii for Philippine seaweed farms

Technique improvement and cost reduction of branch culture, micropropagation, and callus production of carrageenan-yielding seaweeds Kappaphycus alvarezii and Eucheuma denticulatum is presented. Low cost branch culture is possible by enriching seawater with 0.1% coconut water with 1 mg l^–1 indole-3-butyric acid for 24 h wk^–1 or continuous culture with 0.01% Algafer, a Philippine fertilizer. Micropropagation of 0.5 cm explants had almost 100% new branch production demonstrating the viability of callus regenerated plants. The use of carrageenan as a media for callus production was not effective when compared to agar. Propagules of both species, transferred from the University of the Philippine Marine Science Institute (UPMSI) culture facility to the field, showed daily percent growth rates of 5 to 5.5% d^–1 over 84 days. Based on the costs of the UPMSI laboratory, a culture facility in the seaweed farming area is estimated to cost about U. S. $22000 during the initial year and 58% less the second year.     

Alternaria alternata, a new pathotype pathogenic to aphids

A fungus, identified as Alternaria alternata, was isolated from dying or dead aphids and proved to be pathogenic. It was isolated from different parts of Greece from aphid specimens on cultivated plants, ornamentals and weeds. In the laboratory, disease development started with the germination of spores on the insect integument and the subsequent growth of mycelium. The fungus formed apical and intercalary, globose or lobate appressoria which were firmly attached onto the host exoskeleton and facilitated entrance of the mycelium into the insect body. Under favorable conditions of temperature (15–35 °C) and relative humidity (100%), infected aphids died in 2–4 days. A characteristic brown discoloration accompanied the death of the insects. Both mycelial growth and sporulation were profuse on dead specimens. The pathogen infected all 26 aphid species tested but was unable to infect other insects (Drosophila melanogaster and Ceratitis capitata) or aphid host plants. There were significant differences in mortality rate among aphid species only during the first two days after inoculation. It is suggested that A. alternata may be a good candidate to be exploited for the biological control of aphids.     

Revision of the cyprinid genus Placocheilus Wu, 1977 in China,with description of a new species from Yunnan

Placocheilus is re-diagnosed on the basis of oromandibular structures compared to those of closely related groups. It is represented in China by three species restricted to Yunnan, namely P. caudofasciatus from the Tengtiaohe River (Nam Na), P. cryptonemus from the Nujiang Rive (upper Salween River basin), and P. robustus, herein described as a new species from the Yuanjiang River (Red River basin). P. robustus n. sp. is differentiated from its congeners in having a stout caudal peduncle (depth 72.4–82.5% of its length) and the medium-sized scaleless midventral region of belly extended slightly beyond halfway from the pectoral- to pelvic-fin origin. It is further distinguished from P. caudofasciatusin having a coarsely crenulated rostral fold with 18–23 indentations on its distal margin and a smaller mental adhesive disc (length 66.7–71.4% of its width and 37.0–43.1% of the head length).     

Successful inoculation of mature pine with Tricholoma matsutake

Our finding demonstrates, for the first time, that the roots of mature pine trees can be successfully inoculated with a symbiotic ectomycorrhizal fungus, the valuable matsutake mushroom. Long root segments (ca. 5–10 mm in diameter, ca. 50 cm in length) of 50-year-old Pinus densiflora trees were excavated, washed, auxin-treated (2–5 mg indole butyric acid, IBA, per root) and incubated in moist Spagnum moss. Twelve months later, short roots were regenerated, of which approximately 90% were free of mycorrhizae. Mycorrhiza-free short roots were inoculated with mycelial pieces of Tricholoma matsutake and incubated further in a sterilized substrate. Four-and-a-half months later, roots putatively colonized by Matsutake were sampled near the inoculation points. A T. matsutake-specific ITS-rDNA fragment was amplified by nested PCR from approximately 80% of the root samples analyzed, whereas approximately 66% of the root samples processed for staining with Chlorazol black E displayed characteristic T. matsutake Hartig net structures. These results confirm the symbiotic infection of mature P. densiflora roots by matsutake.     

Comparative effects of Glomus mosseae and P fertilizer on foliar mineral composition of Acacia senegal seedlings inoculated with Rhizobium

Summary A factorial experiment with two controlled factors was conducted in the greenhouse with Acacia Senegal seedlings. The substrate was a degraded sandy soil (Dior soil) poor in available P (11 ppm — Olsen). The first controlled factor was soil sterilization, with two levels: (A) sterilized soil; (B) non-sterilized soil. The second factor was fertilization, with six levels: (1) uninoculated control; (2) inoculation with Rhizobium (ORS 1007); (3) inoculation with Glomus mosseae; (4) double inoculation with ORS 1007 and G. mosseae; (5) inoculation with ORS 1007 and 30 ppm phosphorus per plant; (6) inoculation with ORS 1007 and 60 ppm phosphours. The combination of the two factors and their levels led to 12 different plant treatments (A1–A6 and B1–B6). Compared to the control B1, the B5 and B6 treatments containing phosphorus increased: nodule dry weight about 7 times ; leaf dry weight about 4 times ; total N, P and Mg 4–5 times; total K and Ca 3–4 times. The mycorrhizal inoculation had the same positive effect on plant growth and mineral composition but with lower values. Plants inoculated with Rhizobium alone gave the lowest results. The A1 treatment gave lower values than B1. Foliar mineral contents varied within a narrow range (20–30%).