Studies on turfgrass snow mold caused byTyphula ishikariensis. II. Microscopical observation of infected bentgrass leaves

Light and transmission electron microscopy revealed thatTyphula ishikariensis penetrated into bentgrass leaves either through cuticles or stomata either by single hyphae or infection cushions formed on host surfaces. Time course study on infected leaves showed that penetration through stomatal subsidiary cells and their adjacent cells seemed to occur earlier than that through epidermal cells located farther from stomata. More than 30% of epidermal cells were infected by 10 days after inoculation. When hyphae penetrated through an intact cuticle of epidermal cells, they seemed to dissolve host cell walls enzymatically at penetration sites. Physical pressure also seemed to be involved in penetration.     

Flavonol glycosides in the leaves ofTrillium apetalon Makino andT. kamtschaticum pallas

Seven flavonol glycosides were isolated from the leaves ofT. apetalon. They were identified chromatographically and spectrally to be: quercetin/kaempferol 3-O-α-arabinopyranosyl-(1→6)-β-galactopyranoside (TQ and TK), quercetin/kaempferol 3-O-[2‴-O-acetyl-α-arabinopyranosyl]-(1→6)-β-galactopyranoside (TAQ and TAK), quercetin 3-O-β-glucoside (ISQ), isorhamnetin 3-O-α-arabinopyranosyl-(1→6)-β-galactopyranoside (TI) and isorhamnetin 3-O-[2‴-O-acetyl-α-arabinopyranosyl]-(1→6)-β-galactopyranoside (TAI). TQ, TAQ, TI and TAI were major constituents. This is the first report on two new isorhamnetin-type glycosides, TI and TAI. The seven flavonol glycosides identical to those ofT. apetalon were isolated and identified in the leaves ofT. kamtschaticum; TQ and TAQ were also major components, but TI and TAI were only minor components. TI and TAI were not detected in the leaves ofT. tschonoskii. These leaf-flavonoid patterns were discussed from a chemosystematic point of view. Part 3 in the series “Studies of the flavonoids of the genusTrillium”. For Part 2 see Yoshitamaet al., (1997) J. Plant Res.110: 379–381.     

Biological comparisons between arrhenotokous and thelytokous biotypes ofMesochorus nigripes [Hym.: Ichneumonidae]

The host preferences and sex ratios ofMesochorus biotypes from Sweden and Colorado (U.S.A.) were studied in the field and laboratory. TheseMesochorus are secondary parasites ofBathyplectes spp., parasites ofHypera postica (Gyllenhal). The Colorado biotype is 100% thelytokous, and clearly prefersBathyplectes stenostigma (Thomson) as a host. Only an arrhenotokous biotype was positively detected in Sweden, and it appeared to preferB. curculionis (Thomson). However, some of the data suggest that the thelytokous biotype was probably also present in Sweden, at least inB. stenostigma. The behavioral differences and reproductive isolation of the thelytokous and arrhenotokous biotypes indicate that they are separate sibling species, even though they cannot yet be separated morphologically, and isozyme analyses were inconclusive (possibly due to a mixture of biotypes in Sweden).      

Callus proliferation from leaf protoplasts using phenylurea type cytokinin, 4-PU,inBetula platyphylla VAR.Japonica

Summary Protoplasts were isolated from leaves ofBetula platyphylla var.japonica using a 0.6M mannitol solution containing 1% Cellulase Onozuka R-10 and 1% Driselase. The cell division and colony formation were largely enhanced using Murashige and Skoog (1962) liquid medium at half strength (1/2 MS), containing 0.6M mannitol, 0.09M sucrose, and factorial combinations of 0.1–30 μM N-(2-chloro-4-pyridyl)-N′-phenylurea (4-pu) and 0.1–10 μM 1-naphthaleneacetic acid (NAA) or 0.1–30 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The optimal protoplast density was 5–7 × 10⁴/ml. Continuous callus proliferation from protoplasts was achieved by transferring colonies to fresh 1/2 MS agar medium containing 1 μM NAA and 1 μM 4-pu with no mannitol. It appeared that supplementation of the medium with phenylurea type cytokinin, 4-pu gave the successful callus proliferation from the protoplasts ofB. platyphylla.     

Plant regeneration capacity of mesophyll protoplasts from Brassica napus and related species

Protoplasts from a total of thirty-six genotypes of Brassica species – B. napus, B. campestris (syn. B. rapa), B. juncea, and three distant relatives, Orychophragmus violaceus, Isatis indigotica and Xinjiang wild rape – were analysed for shoot regeneration using a feeder culture system. With the exception of B. campestris and Xinjiang wild rape, some genotypes of all the species could regenerate plants with high efficiency (above 20% of isolated calli initiating shoots). Several genotypes with high regeneration ability were elite breeding lines. Culture conditions as well as genotype had a significant impact on shoot regeneration frequency. In particular, silver nitrate added to the regeneration medium at doses of 6 and 30 μM improved shoot regeneration frequency to 25.4% and 52.2% of isolated calli, respectively, compared to 7.3% percent shoot regeneration without silver nitrate in seven responsive genotypes. Addition of silver nitrate to the regeneration medium also induced shoot regeneration in non-responsive genotypes. Intact plants could be obtained within three months from protoplast isolation in the regenerative genotypes using the current culture system. Advantages of mesophyll protoplasts as compared to protoplasts isolated from hypocotyls for genetic manipulation in Brassica species are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Variation within isolates ofTyphula incarnata from localities differing in winter climate

Isolates ofTyphula incarnata, a snow mold fungus, were collected from four localities with different winter climates. Their ecological traits such as mycelial growth rate, sclerotium size, carpogenic germination of sclerotia, and aggressiveness were compared between populations in order to reveal infraspecific differentiation associated with climatic differences. Population variability was evident only in sclerotium germination: isolates from more snowy localities germinated faster than those from less snowy localities.T. incarnata is regarded as a versatile pathogen with no specialized forms in contrast withT. ishikariensis. The germination rate of sclerotia is considered very critical in the life history ofT. incarnata.     

Embryogenic callus induction and plant regeneration media for bentgrasses and annual bluegrass

Summary Embryogenic callus induction and plant regeneration systems have long been established for creeping bentgrass (Agrostis palustris Huds.), but little research has been reported on optimal medium for embryogenic callus induction and plant regeneration in velvet bentgrass (Agrostis canina L.), colonial bentgrass (Agrostis capillaries L.), and annual bluegrass (Poa annua L.). The present study compared 14 callus induction media and eight regeneration media for their efficacies on embryogenic callus induction and plant regeneration in these four species. The embryogenic callus initiation media contained the Murashige and Skoog inorganic salts and vitamins supplemented with 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-anisic acid and 6-benzyladenine. l-Proline or casein hydrolyzate was included in some media to stimulate embryogenic callus formation and plant regeneration. The frequencies of embryogenic callus formation ranged from 0% to 38% and exhibited medium differences within each of the four species. Callus induction media, plant regeneration media, and genotypes affected plant regeneration rates, which varied between 0% and 100%. The embryogenic callus induced on Murashige and Skoog medium supplemented with 500 mgl⁻¹ casein hydrolyzate, 6.63 mg l⁻¹ (30 μM) 3,6-dichloro-anisic acid and 0.5–2.0 mg l⁻¹ (2–9 μM) 6-benzyladenine had much higher regeneration rates than those formed on other callus induction media. Embryogenic callus of annual bluegrass had higher regeneration rates than those of bentgrass species. MSA2D, a media containing 2 mgl⁻¹ (8 μM) 2,4-dichlorophenoxyacetic acid, 100 mgl⁻¹ myo-inositol, and 150 mgl⁻¹ asparagine, was effective in promoting embryogenic callus formation in creeping bentgrass but not in colonial and velvet bentgrasses and annual bluegrass.     

Morphological variation inBemisia endosymbionts

Summary The ultrastructure of the endosymbionts of several populations of whitefly (Homoptera: Aleyrodidae) was examined using transmission electron microscopy. Consistent differences in morphology and relative number of endosymbionts were observed between species and biotypes of whitefly within the Bemisia taxon.Bemisia argentifolii (=B. tabaci B biotype) individuals from Hawaii, Florida, and Arizona contained two morphological types of microorganisms housed within the mycetocyte cells of immature whiteflies. In contrast, individuals from populations ofB. tabaci A biotype from Arizona and Mexico, andB. tabaci Jatropha biotype from Puerto Rico, consistently contained three distinct morphological types of microorganisms within their mycetocytes. Organisms fromB. tabaci A and Jatropha biotypes differed from each other in the relative frequency of each type of microorganism. These observations suggest that different whitefly biotypes may have variable combinations of micro-fauna, with some possibly unique to each group, and furthers the hypothesis that variation in whitefly endosymbionts may be associated with the development of biotypes.     

Rearing of two predators,Thanasimus dubius andTemnochila virescens, for the biological control ofIps grandicollis in Australia

Rearing methods for two coleopterous predators,Thanasimus dubius andTemnochila virescens, imported into Australia for the biological control ofIps grandicollis, were developed. Bionomic data obtained from laboratory rearings between 1982–1987 showed thatT. dubius eggs took about 7 days to hatch and that duration of the larval stage was about 42 days. Observations showed thatT. dubius had a prolonged prepupal stage (x=56.4 days, range 14–274 days), which was probably non-diapausal in nature. Mean adult longevity was 50 days (range 1–358 days).Temnochila virescens eggs took almost 9 days to hatch, and a lengthy larval stage (x=155.4 days, range 73–333 days) was observed. Mean duration of the pupal stage was 14 days (range 7–28 days). A long preoviposition period (x=141 days, range 47–206 days) was observed, and adults were very long-lived (x=232.7 days, range 14–667 days). Capacity for increase (r_c) calculated from rearing data suggested that numbers ofT. dubius could be increased faster thanT. virescens. Mortality between 1982–1987 averaged about 70% for both species. However, mortality ofT. dubius in 1987 increased significantly, suggesting that inbreeding or other methodological factors could be responsible. A mass-rearing method usingIps-infested pine billets was developed as a cheaper alternative to laboratory rearing, and was shown to be effective in producing large numbers of insects for release.     

Electrophoretic karyotyping and antifungal susceptibility patterns of Candida parapsilosis clinical isolates causing deep and superficial fungal infections

Forty-six isolates of Candida parapsilosis, each from a single patient, were collected from July 1993 through March 1999 at the University of Ancona Hospitals and Clinics. Twenty-eight strains were isolated from superficial lesioned sites, including skin, nails and other sources while 18 strains were isolated from blood. The isolates were typed by electrophoretickaryotyping (EK) and tested for their susceptibility to fluconazole (FLC),itraconazole (ITC), flucytosine (5-FC), and amphotericin B (AMB). Ourdata confirmed that EK is a useful technique for DNA typing of isolates ofCandida parapsilosis and showed that the source of isolation is notassociated with a given DNA type. Although strains belonging to this speciesof Candida are susceptible to the most common antifungals, including the triazoles, the degree of ITC susceptibility was dose dependent (MIC rangingfrom 0.25–0.5 μg/ml) for 98% of the isolates. This revised version was published online in June 2006 with corrections to the Cover Date.     

Further studies on the surface charge of various strains ofTrichomonas vaginalis andTritrichomonas foetus

The surface charge of three strains ofTrichomonas vaginalis and five strains ofTritrichomonas foetus was determined by direct measurement of the mean cellular electrophoretic mobility (EPM) of cells suspended in solutions of different ionic strength and pH. No differences were observed in the mean EPM among the two species, although significant differences among the strains exist. Strains that are more pathogenic to mouse, as measured using the subcutaneous assay, had a surface more negative. Treatment of the parasites with trypsin or neuraminidase reduced significantly their mean EPM and increased their isoelectric point.Tritrichomonas foetus was more sensitive to the enzyme treatment thanT. vaginalis. Enzyme-treated cells recovered their normal EPM if, after enzyme treatment, they were incubated in fresh culture medium. The recovery process of trypsintreated cells was inhibited 10–20% by addition of inhibitors of either protein synthesis (puromycin) orN-glycosylation of proteins (tunicamycin) to the incubation medium, suggesting that a cytoplasmic pool of sialoglycoproteins may exist. The recovering of the EPM ofT. foetus andT. vaginalis previously treated with neuraminidase was inhibited by puromycin or tunicamycin about 40–50% and 17–30%, respectively. These observations suggest that sialoglycolipids exist on the surface of both parasite species, and that they contribute more to the surface charge ofT. vaginalis than to that ofT. foetus.     

Purification of cationic peroxidases bound ionically to the cell walls from the roots ofZinnia elegans

Cell wall-bound and tracheary element-specific peroxidase isoenzymes, designated P5A and P5B, were shown previously to be associated with lignification during the differentiation into tracheary elements of single cells isolated from the mesophyll ofZinnia elegans (Satoet al. Planta 189: 584–589, 1993; Planta 196: 141–147, 1995). Isoenzymes corresponding to P5 (RP5A and RP5B) were present at a relatively high level in the roots ofZinnia elegans. These isoenzymes were purified from theZinnia roots by several column-chromatographic steps. Both RP5A and RP5B had molecular masses of 35 kDa. Purified RP5A and RP5B were cleaved by CNBr and the partial amino acid sequences of these isoenzymes were determined.     

Materials forPythium flora of Japan VIII Two species ofPythium:P. pyrilobum andP. oligandrum

Two species ofPythium (P. pyrilobum, P. oligandrum) having spherical sporangia with complex subglobose elements were isolated from the crown of creeping bentgrass [Agrostis palustris (cv. Penncross)] and from vegetable field soil, respectively. They are reported for the first time in Japan.     

Reproduction and larval development of the gastropodAlaba vladivostokensis in Vostok Bay, the Sea of Japan

The reproduction and development of the prosobranch gastropodAlaba vladivostokensis Bartsch were studied. In Vostok Bay,A. vladivostokensis reproduces in the warmest season at water temperatures of 19–23°C. The females lay egg masses in the shape of flat, coiled bands on eelgrass leaves and on sargosso thalluses. The total period of development from egg laying to larval settling takes 25–27 days. The reproduction and development patterns and larval morphology ofA. vladivostokensis are similar to that ofAustralaba picta (A. Adams), which inhabits the coast of Japan. It is suggested that the population ofA. vladivostokensis in Peter the Great Bay should considered as part of the wide geographical range ofA. picta.     

Contribution ofAzospirillum brasilense to the nitrogen needs of grain sorghum (Sorghum bicolor (L) Moench) in humid sub-tropics

Summary Field experiments were conducted to assess the contribution ofAzospirillum brasilense to the N needs of grain sorghumcv. CSH — 5 during monsoon (June–October) seasons of 1978 and 1979.A. brasilense contributed to the N uptake by crop in the range from 5.8 to 19.6 kg N/ha. However, the contribution ofA. brasilense to the N needs of sorghum was more when sorghum was manured with farmyard manure at the rate of 10 tons/ha. Publication of G.B.P.U.A.T. Expt. Station, Pantnagar — 263145, India.     

Isolation ofTricholoma matsutake andT. bakamatsutake cultures from field-collected ectomycorrhizas

Tricholoma matsutake was isolated into pure cultures from field samples of ectomycorrhizas onPinus densiflora. The mycorrhizal tips were collected at different times of the year from a colony ofT. matsutake in aP. densiflora stand. The mycorrhizal tips were continuously washed with sterilized distilled water and diluted Tween 80 solution, surface-sterilized with calcium hypochlorite solution, and inoculated on several kinds of nutrient agar media. Most of the mycorrhizal tips collected in winter and spring produced colonies that were morphologically similar to cultures ofT. matsutake isolated from basidiocarps. The identity of isolates obtained from mycorrhizas was further confirmed to beT. matsutake based on fungal morphology and RFLP patterns of PCR amplified rDNA. The feasibility ofT. bakamatsutake isolation into pure culture from ectomycorrhizas onQuercus serrata was also confirmed. These results indicated that mycelium of matsutake mushrooms can be isolated into pure culture from ectomycorrhizas at different times of the year. Mycorrhizas of bothT. matsutake andT. bakamatsutake were not observed to have any specific association with soil fungi such asMortierella spp.     

The influence of natural enemies on brood production inIps typographus (Col. scolytidae) with special reference to egg-laying and predation byThanasimus formicarius (Col.: Cleridae)

The predatorThanasimus formicarius (L.) (Coleoptera, Cleridae) and its preyIps typographus (L.) (Coleoptera, Scolytidae) were studied in the laboratory and the field. In the laboratory, 11T. formicarius laid 71–132 eggs (mean=162) during 66–123 days. During this time they ate 66–132I. typographus adults per pair (male +female). The number of eggs laid per female was not correlated with life span or the number ofIps eaten. In the field, predation byT. formicarius larvae and other natural enemies onI. typographus brood was studied in the last year of an outbreak. Caged and uncaged spruce bolts attacked byI. typographus were used, and pairs ofT. formicarius were released in the cages. The treatments were: uncaged bolts, caged bolts withoutT. formicarius, caged bolts with 4T. formicarius pairs, and caged bolts with 8T. formicarius pairs. The productivity ofI. typographus was highest in the caged bolts withoutT. formicarius (mean=4.5 offspring/female) and lowest in the uncaged bolts (mean=0.9 offspring/female). The density ofI. typographus galleries was similar in the different treatments. Hence, the variation in productivity between treatments could not have been due to differences in the levels of intraspecific competition. There was no difference in bark beetle productivity or density ofT. formicarius larvae between bolts with 4 pairs ofT. formicarius and bolts with 8 pairs (mean=2.5 offspring/female). This indicates that some kind of interference occurred betweenT. formicarius individuals (e.g. cannibalism) and that a maximum level of predation was reached. Predation by larvae ofMedetera spp. (Diptera, Dolichopodidae),Thanasimus spp. and other beetles, and parasitism by wasps (Hymenoptera, Pteromalidae) probably caused the low productivity in the uncaged bolts.      

Bionomics ofTetrastichus sesamiae [Hymenoptera: Eulophidae], a pupal endo-parasitoid ofMaruca testulalis [Lepidoptera: Pyralidae]

The biology and behaviour ofTetrastichus sesamiae Risbec, a pupal endoparasitoid ofMaruca testulalis Geyer, were studied under laboratory conditions. Most adults emerged from the host pupae between 08.00 h and 09.00 h and mating and oviposition started almost immediately. Both ♂♂ and ♀♀ mated repeatedly, and each ♀ could lay eggs for up to 6 days and in up to 5 host pupae. Progeny production ranged from 0–263 offspring per ♀ and adult longevity was from 4.3–13.9 days. The quality of food available to the adults was a major factor influencing progeny production, and longevity. The species was capable of parasitizing and completing development on pupae of such other major Lepidopteran crop pests asChilo partellus Swinhoe,Busseola fusca Fuller,Eldana saccharina Wlk andSpodoptera exempta Wlk, an important finding for biological control of these pests under intercropped agro ecosystems. Apart from parasitism, ♀♀ ofT. sesamiae also caused considerable mortality by stinging, and presumably paralyzing, host pupae without ovipositing.      

Herbicide-resistant transgenic creeping bentgrass plants obtained by electroporation using an altered buffer

Modification of an electroporation buffer using Ca(NO₃)₂ and elevated pH (9–10) appeared to have a favorable effect on gene transfer to creeping bentgrass (Agrostis palustris Huds.) Penncross protoplasts, resulting in an increase in the transformation frequency of about twofold. Following electroporation with the plasmid pARK22 containing the bar gene, a total of 278 bialaphos-resistant cell colonies were obtained from four experiments. The bialaphos-resistant regenerants proved to be transgenic by Southern hybridization of the amplified DNA. All the tested transgenic plants showed herbicide (HERBIE) resistance at the field rate of 0.5–1% (vol/vol). Ammonia contents in leaves after spraying with the herbicide increased less in transgenic plants than in untransformed control plants. Received: 10 February 1998 / Revision received: 8 April 1998 / Accepted: 24 April 1998