In vitro and in vivo host range of Anticarsia Gemmatalis multiple nuclear polyhedrosis virus

Summary A clone of the wild type (wt) Anticarsia gemmatalis multiple nuclear polyhedrosis virus AgMNPV, derived from a geographical isolate (Hondrina, Brazil) and designated AgMNPV-CL4-3A1, was used to determine the host range of this virus in six established lepidopteran cell lines: Anticarsia gemmatalis (BCIRL-AG-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Heliothis virescens (BCIRL-HV-AM1), Helicoverpa armigera (BCIRL-HA-AM1), Trichoplusia ni (TN-CL1), Bombyx mori (BMN), and a coleopteran cell line Anthonomus grandis (BRL-AG-1). In addition, the in vivo host range of this clone was also assayed in larvae of Helicoverpa zea, Heliothis virescens, Trichoplusia ni, and the homologous species Anticarsia gemmatalis by probit analysis. On the basis of temporal studies of TCID₅₀ values, BCIRL-HV-AM1 cells gave the highest extracellular virus (ECV) titer (9.7×10⁶ TCID₅₀/ml) followed by BCIRL-HA-AM1 cells (8.3×10⁵ TCID₅₀/ml) and BCIRL-AG-AM1 cells (3.2×10⁵ TCID₅₀/ml). In addition, a low ECV titer of 1.37×10³ TCID₅₀/ml was detected from TN-CL1 cells 96 h postinoculation, while BRL-AG-1, BMN, and BCIRL-HZ-AM1 cells were nonpermissive to AgMNPV-CL4-3A1 on the basis of TCID₅₀ results. AgMNPV-CL4-3A1 and the wild type AgMNPV had similar restriction profiles that were different from wild type AcMNPV. The LC₅₀ values were 96.9, 564.6, 733.3, and 1.1×10⁴ occlusion bodies/cm² of diet for A. gemmatalis, Helicoverpa zea, Heliothis virescens, and T. ni, respectively. This article presents the results of research only. Mention of proprietary products in this article does not indicate endorsement or a recommendation for use by USDA-ARS. All programs and services of the USDA are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, marital status or handicap.     

Studies in parasitic watermolds of Kumaun Himalayas: The host range of five species of olpidiopsis

The host range of five species of Olpidiopsis in some species of Achlya, Aphanomyces, Brevilegnia, Isoachlya, Saprolegnia, Thraustotheca and Pythium was studied. Species were different in their host range.     

Broad host range fluorescence and bioluminescence expression vectors for Gram-negative bacteria

Tagging of bacteria with living colors and living light allows increasingly valuable new imaging and detection technologies to be accessible to researchers. In this study, we aimed to create stable broad host range expression vectors for tagging Gram-negative bacteria with fluorescence and bioluminescence. To accomplish this, a mutated form of promoterless green fluorescent protein (gfp) gene, gfpmut3a, from Aequorea victoria and promoterless bacterial luciferase genes, luxCDABE, from Photorhabdus luminescens were inserted into broad host range plasmid pBBR1MCS4. Expression of gfp and luxCDABE genes was driven by lacZ promoter. In addition, dual versions with both gfpmut3a and luxCDABE genes and inducible versions carrying lacI(q) gene were also constructed. These new broad host range vectors containing a stable broad host range origin of replication and mobility genes can be transferred to Gram-negative bacteria by either electroporation or conjugal mating and maintained stably. Availability of these expression vectors should be useful in developing new approaches to study a broad variety of Gram-negative bacteria, particularly for applications investigating host-pathogen interactions in vivo and in vitro.     

Studies on Chlorella protoplasts

Protoplasts of Chlorella saccharophila (Krüger) Nadson were obtained by cellulase digestion of the microfibrillar inner compount of the cell wall after the resistant outermost layer had been scratched with sea sand. The absence of the cell wall was demonstrated immunologically, electron microscopically and by staining, thus confirming the protoplastic nature of the treated cells. After transfer to an enzyme-free medium regeneration of a thin cell wall was observed. The regeneration of the cell wall obviously followed the same steps as does the cell wall development of the autospores. At least 50% of the protoplasts were able to form colonies when plated on a suitable agar medium.     

Phage sensitivity and host range of Rhizobium strains isolated from root nodules of temperate legumes

Twenty-five Rhizobium strains were isolated from root nodules of Astragalus spp. (10), Hedysarum alpinum (7), Glycyrrhiza pallidiflora (3) and Ononis arvensis (5). The sensitivity of these strains to bacteriophages of Rhizobium loti, R. meliloti, R. galegae and R. leguminosarum was studied. Phages specific to R. loti strains were shown to induce the phage lysis of several Astragalus, Hedysarum and Ononis rhizobia. Ten R. loti strains tested for nodulation abilities on the plant hosts under investigation were able to develop nitrogen-fixing nodules on the Ononis arvensis roots. On the other hand, rhizobia from Ononis and Glycyrrhiza could form an effective symbiosis with Lotus corniculatus plants, so these bacteria are considered to belong to the Rhizobium loti taxon. Bacterial strains isolated from Astragalus and Hedysarum were observed to cross-nodulate their plant hosts as well as Oxytropis campestris, Glycyrrhiza uralensis and Ononis arvensis plants, whereas they could not nodulate Lotus plants. It is concluded that these Rhizobium strains comprise a cross-inoculation group related to Rhizobium loti. ei]{gnR O D}{fnDixon}     

Studies on the ultrastructure and taxonomy of the genusTetraselmis (Prasinophyceae)

Ultrastructural investigations on many isolates ofTetraselmis have revealed that the species have characteristic fine structural features of the pyrenoid and it was proposed (Horiet al., 1982) that the genus be subdivided into four subgenera. In the present study of this series, species of the subgenusPrasinocladia, includingTetraselmis marina, T. verrucosa andT. verrucosa f.rubens, are described in detail.     

The ultrastructure of the outer cell wall-layer ofChlorella mutants with and without sporopollenin

Chlorella fusca wild type strain C-1.1.10 is able to synthesize ketocarotenoids (KC) and sporopollenin (SP) which are present in the cell wall (CW) as integral components. Mutants derived from this strain are unable to synthesize KC and SP. The CW ultrastructure of both, wild type and mutants, was investigated by TEM after different staining procedures. Strains containing SP in the CW exhibit a trilaminar structure of the outer layer of the CW, while mutants defective in KC- and SP-synthesis differ in this respect. Fragments of unit membranes, various membranous vesicles and structures often are found in the space between the CW and the plasmalemma; this perhaps indicates some steps of CW- and sporopollenin-synthesis. These findings again strongly confirm the close connection between the synthesis of KC, SP, and the trilaminar structure of the outer CW-layer.     

Studies on the ultrastructure and taxonomy of the genusTetraselmis (Prasinophyceae)

Comparative ultrastructural investigations on many isolates ofTetraselmis from Japan and the Pacific coast of North America, and on cultures from the Culture Centre of Algae and Protozoa, Cambridge, England, have revealed that the species have characteristic fine structural features of the pyrenoid. Using the pyrenoid structure as a basic character it is proposed that the genus be subdivided into four subgenera,Tetraselmis, Prasinocladia, Tetrathele andParviselmis. In the present paper, species of the subgenusTetraselmis, includingT. cordiformis, T. ascus, T. convolutae andT. astigmatica sp. nov., are described in detail.     

Population growth of some genera of cladocerans (Cladocera) in relation to algal food (Chlorella vulgaris) levels

We studied the patterns of population growth of 7 cladoceran species (Alona rectangula, Ceriodaphnia dubia, Daphnia laevis, Diaphanosoma brachyurum, Moina macrocopa, Scapholeberis kingi and Simocephalus vetulus) using 6 algal densities, viz. 0.05×10⁶, 0.1×10⁶, 0.2×10⁶, 0.4×10⁶, 0.8×10⁶ and 1.6×10⁶ cells ml^–1, of Chlorella vulgaris for 18 – 30 days. In terms of carbon content these algal concentrations corresponded to 0.29, 0.58, 1.16, 2.33, 4.65 and 9.31 g ml^–1, respectively. Cladocerans in the tested range of algal levels responded similarly, in that increasing the food concentrations resulted in higher numerical abundance and population growth rates (r). The peak population densities were (mean±standard error) 71±5; 17.1±0.4, 3.6±0.3, 12.7±1.1, 18.2±2.7, 15.8±1.0 and 10.9±0.02 ind. ml^–1, respectively for A. rectangula, C. dubia, D. laevis, D. brachyurum, M. macrocopa, S. kingi and S. vetulus. In general, the lowest r values were obtained for D. laevis (0.01±0.001) at 0.05×10⁶ cells ml^–1 food level while the highest was 0.283±0.004 for A. rectangula at 1.6×10⁶ cells ml^–1 of Chlorella. When the data of peak population density for each cladoceran species were plotted against the body length, we found an inverse relation, broadly curvilinear in shape. From regression equations between the food level and rate of population increase, we calculated the theoretical food quantity (the threshold level) required to maintain a zero population growth (r = 0) for each cladoceran species, which varied from 0.107 to 0.289 g ml^–1 d^–1 depending on the body size. When we plotted the cladoceran body size against the corresponding threshold food levels, we obtained a normal distribution curve. From this it became evident that for up to 1300 m body size, the threshold food level increased with increasing body size; however, beyond this, the threshold level decreased supporting earlier observations on rotifers and large cladocerans.     

Laboratory biology and host range ofHydrellia balciunasi [Diptera: Ephydridae]

Hydrellia balciunasi Bock, a native of Australia, was evaluated in quarantine in Florida, USA, for its potential as a biocontrol agent of the submersed aquatic weed,Hydrilla verticillata (L.f) Royle. Larvae are leafminers. Mean total development time at 27°C was 22.8 days. Mean duration of the egg stage was 3.0 days, larval was 11.5 days, and puparial was 8.3 days. Mean fecundity was 35.5 eggs. Mean female longevity was 19.7 days, and mean male longevity was 15.6 days. The sex ratio was 1.1∶1 (male: female). Fourteen plant species closely related to hydrilla in 4 families plus rice were tested in no-choice larval development tests and an additional 27 plant species in 16 families were tested in multi-choice tests. Larvae mined in 2 test plant species,Potamogeton pusillus L. andP. crispus L., but developed (1%) only on the introduced weedP. crispus L. Females oviposited on most test plants. Permission to release this fly in the United States was received from federal and state (Florida) officials, and it was released from quarantine on 24 May 1989.      

The host range and biology of the mesquite psyllid Heteropsylla texana

Prosopis spp. areintroduced rangeland weeds in Australia. In asearch for biological control agents, thepsyllid Heteropsylla texana, which causessevere distortion to growing leaf and floralshoots, was imported from Texas, USA forhost range testing in quarantine facilities.No-choice tests were conducted on 60 plantspecies including P. pallida, P. velutinaand P. juliflora. In these trials, adultssurvived on 45 non-target plants but ovipositedonly on the Prosopis spp., Dichrostachys spicata and Acaciabidwillii. Nymphs developed to ovipositingadults on all Prosopis spp. and both D. spicata and A. bidwillii. Developmentof small numbers of adults on D. spicataand A. bidwillii occurred for only onegeneration. Damage was noticed only on Prosopis spp. In multiple-choice trials usingthree plant species, oviposition and subsequentdevelopment of eggs and nymphs to adults tookplace on P. pallida and D. spicatabut not on A. bidwillii. The low numbersdeveloping, the longer development times toadults when reared on D. spicata and A.bidwillii, and the failure of these plantsto sustain populations beyond one generationindicate that these plants are not hosts ofH. texana. While adult survival on manytest plants may imply that adult feedingoccurred, the risk to populations of theseplants in the field is negligible. It wasconcluded that H. texana is specific toProsopis spp. and could be released inAustralia for control of Prosopis spp.Observations of the biology of this speciesmade during the course of rearing andexperimentation indicated that nymphs developedthrough five instars to adults in 7–8 days sothat total development from egg to adult takes13–17 days. Females produce up to 100 eggs.     

The endosymbiotic unit ofClimacostomum virens andChlorella sp. I. Morphological and physiological studies on the algal partner and its localization in the host cell

Summary The ciliateClimacostomum virens forms an endosymbiotic association with coccoid chlorophycean algae which can be isolated and grown as sterile mass cultures with an inorganic medium. According to both morphological and physiological properties, the algae probably belong to the genusChlorella and have some features in common with symbiotic chlorellae isolated from the ciliatesParamecium bursaria andStentor polymorphus. These and other endosymbiotic chlorellae studied so far excrete maltose or glucose, but algae fromClimacostomum virens show a different excretion pattern by releasing glucose, fructose and xylose. Possible biosynthetic pathways are discussed. Algae inClimacostomum virens are either located individually in special perialgal vacuoles where they are probably protected against attack by host lytic enzymes or to a lesser extent in food vacuoles in different states of digestion. The endosymbiotic character of theClimacostomum virens-Chlorella sp.-association is discussed.Dedicated to Prof. H. A.von Stosch on the occasion of his 75th birthday.     

Structural genesvirC1 andvirC2 of the host range determiningvirC operon are determinants of virulence inAgrobacterium tumefaciens

The host range determiningvir C operon ofAgrobacterium tumefaciens is known to consist of two open rea’ding frames designatedvirC1 andvirC2. Earlier work that employed insertional mutations invirC1 andvirC2 established the role of thevirC2 component in the determination of virulence. In this work a plasmid with an internal deletion invirCl was constructed. This deletion derivative restored virulence to bacteria carrying a mutation in thevirC2 region but not to bacteria carrying avirC1 mutation. This evidence establishes that bothvirC1 andvirC2 are required for efficient host plant transformation byAgrobacterium tumefaciens.     

Alatae production and population increase of aphid vectors on virus-infected host plants

Summary Zucchini yellow mosaic virus (ZYMV) and Aphis gossypii Glover are two components of a recently identified plant-parasite system that provides an excellent opportunity to study interrelations between a virus and a vector that share the same host, but have no direct physiological interaction. In a field experiment we documented numbers of alate and apterous A. gossypii on healthy Cucurbita pepo and on plants inoculated with virus 0, 7, 14, and 21 days before aphid infestation. When plants were inoculated and infested simultaneously, more than twice as many alatae were produced after 20 days of colony growth than on any other treatment. This indicates that properties unique to the early stages of viral infection somehow stimulated wing formation. Because it is spread by the activities of alatae, virus dispersal would be greater as a result of these properties. Developmental rate, total numbers of aphids, and numbers of alatae and apterae decreased as the time between virus inoculation and aphid colonization increased.     

The host range and selectivity of a parasitic plant: Rhinanthus minor L.

Summary Rhinanthus minor (Yellow-rattle) is a widespread hemiparasitic plant of grassland habitats throughout Britain. Association analysis of the dune vegetation at Holme-next-the-Sea in eastern England revealed only two potential host plants through positive association. In contrast direct examination of the root systems revealed haustorial connections with 20 host species. The number of species parasitized by one plant ranged from one to seven. Data from another four sites in Britain and one in central Europe indicate that the natural host range of R. minor encompasses at least 50 species from 18 families with 22% in the Leguminosae and 30% in the Gramineae. Comparison of the number of haustorial connections made to each species with the abundance of roots in the soil shows that R. minor is a highly selective parasite, but that the selectivity is not consistent between populations or between plants from different parts of the same population. The reasons for host selectivity are discussed.     

Restriction site map of the Chlorella virus PBCV-1 genome

The virus PBCV-1, which replicates in a Chlorella-like green alga, has a dsDNA genome. The DNA was mapped for BamHI, HindIII, and PstI restriction sites. The resulting map has a size of 333 kbp and is circular—indicating either covalently closed circular DNA or circularly permuted linear DNA. Several regions of repetitive DNA were also identified and located on the restriction map.     

Symbiotic Chlorella sp. of the ciliate Paramecium bursaria do not prevent acidification and lysosomal fusion of host digestive vacuoles during infection

Summary. Each symbiotic Chlorella sp. of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole, and thereby the alga is protected from digestion by lysosomal fusion. Algae-free cells can be reinfected with algae isolated from algae-bearing cells by ingestion into digestive vacuoles. To examine the timing of acidification and lysosomal fusion of the digestive vacuoles and of algal escape from the digestive vacuole, algae-free cells were mixed with isolated algae or yeast cells stained with pH indicator dyes at 25 ± 1 °C for 1.5 min, washed, chased, and fixed at various time points. Acidification of the vacuoles and digestion of Chlorella sp. began at 0.5 and 2 min after mixing, respectively. All single green Chlorella sp. that had been present in the host cytoplasm before 0.5 h after mixing were digested by 0.5 h. At 1 h after mixing, however, single green algae reappeared in the host cytoplasm, arising from those digestive vacuoles containing both nondigested and partially digested algae, and the percentage of such cells increased to about 40% at 3 h. At 48 h, the single green algae began to multiply by cell division, indicating that these algae had succeeded in establishing endosymbiosis. In contrast to previously published studies, our data show that an alga can successfully escape from the host’s digestive vacuole after acidosomal and lysosomal fusion with the vacuole has occurred, in order to produce endosymbiosis. Correspondence and reprints: Biological Institute, Faculty of Science, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan.     

Diversity, host, and habitat specificity of oomycete communities in declining reed stands (Phragmites australis) of a large freshwater lake

We studied the diversity of oomycetes in a declining reed belt (Phragmites australis) of Lake Constance, Germany, using conventional baiting with specific reed and standard oak baits, accompanied by molecular techniques. Apart from an Aphanomyces sp. and a Phytophthora sp., baiting from reed rhizosphere samples from flooded, as well as drier, littoral sites revealed only Pythium spp. A total of 67 oomycete isolates was classified according to PCR-RFLP banding patterns and ITS sequencing, and 18 different sequence types could be separated. The majority of these seemed previously unknown species, as indicated by the degree of similarity to those deposited in nucleotide databases. Species communities in both flooded and drier habitats or both reed and oak baits clearly differed from one another, and only few species occurred in both dry and flooded sites, or in both oak and reed baits. A frequently occurring group of related Pythium species appeared to be specifically associated with reed, and these were the only species that proved pathogenic towards this host in vitro. Our study proved that unexplored natural ecosystems harbour diverse communities of oomycete species with specific habitat and host preferences within close-by, but ecologically contrasting, sites. Among the species isolated, those associated with the predominating plant might accumulate and thus may be reed pathogens of considerable importance.     

Flocculation of algae using chitosan

Flocculation of three freshwater algae, Spirulina,Oscillatoria and Chlorella, and onebrackish alga, Synechocystis, using chitosan was studiedinthe pH range 4 to 9, and chlorophyll-a concentrations inthe range 80 to 800 mg m^–3, which produces aturbidity of 10 to 100 nephelometric turbidity units (NTU) in water. Chitosanreduced the algal content effectively by flocculation and settling. Theflocculation efficiency is very sensitive to pH, and reached a maximum at pH7.0for the freshwater species, but lower for the marine species. The optimalchitosan concentration that is required to effect maximum flocculation dependedon the concentration of alga. Flocculation and settling were faster whenconcentrations of chitosan higher than optimal are used. The settled algalcellsare intact and live, but will not be redispersed by mechanical agitation. Thede-algated water may be reused to produce fresh cultures of algae.     

Structural variability in the genome of the Thermoproteus tenax virus TTV1

Summary Six variants of the TTV1 genome, including the primary isolate, have been characterized. DNA sequence comparison of wild-type virus (WT) and one of the variants (VT3) showed that differences are due to insertions and deletions that were confined to contiguous portions of two distinctClaI fragments. Seven similar short DNA sequences (30–102 bp) were involved in the variation. The deletions and insertions of these short DNA sequences occurred in every case adjacent to the 8 by consensus sequence 5-ACXCCTAC-3 which formed the 5 flank of the segments involved.