Zealand Isolates Research Articles

Cell morphology and PSP‐toxin profiles of Alexandrium minutum in the Marlborough Sounds, New Zealand

Thecal plate morphology and paralytic shellfish poisoning (PSP) toxin spectra produced by three strains of a small Alexandrium species isolated from the Marlborough Sounds, New Zealand were examined. Although these cells displayed minor morphological features described in the literature as characteristic of both A. minutum and A. lusitanicum, the former designation was adopted. High performance liquid chromatography (HPLC) analysis of culture extracts showed the toxin profiles to consist predominantly of various proportions of the carbamate toxins GTX1, GTX2, GTX4, neoSTX, and STX. These toxin profiles were similar to those observed in other New Zealand isolates of A. minutum. They were however rather different from those observed in this species elsewhere in the world though the specific toxicity of these cells (1.8–2.2 pg STX equivalents per cell) was similarly low.

Relationships among Colletotrichum isolates from fruit-rots assessed using rDNA sequences

Isolates of Colletotrichum associated with fruit rots in New Zealand were used to test the applicability of morphological, cultural, and rDNA sequence data to clarify relationships within this genus. Morphological and cultural features were used to recognize 16 distinct groups among the New Zealand fruit-rotting isolates. These included the currently accepted species C. coccodes, C. musae, C. orbiculare, and Glomerella miyabeana as well as 3 C. acutatum like groups and 9 C. gloeosporioides-like groups. Analysis of rDNA sequences from the D2 region allowed the isolates to be separated into 4 broad groups (the relationships of C. coccodes remaining unresolved), the various morphological/cultural groups clustering as subgroups among the rDNA sequence groups. The same 4 groups were recognized when rDNA sequences from the New Zealand isolates were compared with those from Colletotrichum isolates from other hosts and other countries. Taxonomic treatment of the molecular and morphological/cultural data is discussed. A trinomial group-species concept may provide the best approach to subgeneric classification within Colletotrichum. The species name (based on rDNA sequence similarity and supporting morphological and cultural features) indicates the broad group within Colletotrichum to which the isolate belongs; the subspecific name provides additional information on host specialisation, morphological variation, etc. However, before the utility of this concept can be properly assessed, the evolutionary and genetic relationships between the various morphological and sequence-defined groups need to be better understood. If such a scheme is shown to be practical in a biological sense then a number of taxonomic and nomenclatural problems will need to be resolved before it could be adopted.

Relationships among Colletotrichum Isolates from Fruit-Rots Assessed Using rDNA Sequences

Isolates of Colletotrichum associated with fruit rots in New Zealand were used to test the applicability of morphological, cultural, and rDNA sequence data to clarify relationships within this genus. Morphological and cultural features were used to recognize 16 distinct groups among the New Zealand fruit-rotting isolates. These included the currently accepted species C. coccodes, C. musae, C. orbiculare, and Glomerella miyabeana as well as 3 C. acutatum like groups and 9 C. gloeosporioides-like groups. Analysis of rDNA sequences from the D2 region allowed the isolates to be separated into 4 broad groups (the relationships of C. coccodes remaining unresolved), the various morphological/cultural groups clustering as subgroups among the rDNA sequence groups. The same 4 groups were recognized when rDNA sequences from the New Zealand isolates were compared with those from Colletotrichum isolates from other hosts and other countries. Taxonomic treatment of the molecular and morphological/cultural data is discussed. A trinomial group-species concept may provide the best approach to subgeneric classification within Colletotrichum. The species name (based on rDNA sequence similarity and supporting morphological and cultural features) indicates the broad group within Colletotrichum to which the isolate belongs; the subspecific name provides additional information on host specialisation, morphological variation, etc. However, before the utility of this concept can be properly assessed, the evolutionary and genetic relationships between the various morphological and sequence-defined groups need to be better understood. If such a scheme is shown to be practical in a biological sense then a number of taxonomic and nomenclatural problems will need to be resolved before it could be adopted.

Biocontrol of Phytophthora cactorum in vitro with Enterobacter aerogenes

Phytophthora cactorum causes significant mortality of mature trees in New Zealand pipfruit orchards. Replants of infected sites often succumb to this pathogen within 3 years. Enterobacter aerogenes, strain B8 was reported to control P. cactorum infection of apples in nursery soils in British Columbia. This strain was imported to New Zealand in 1991 and compared with indigenous isolates of E. aerogenes. The Canadian isolate differed from New Zealand isolates in utilisation of tartrate, tolerance of NaCl (5 cf. 7%), and inhibition of P. cactorum in vitro. Strain B8 was fungicidal whereas New Zealand isolates were fungistatic. Electron microscopy of mycelium exposed to strain B8 on agar revealed extensive disruption of cell membranes, intermixing and aggregation of organelles, necrosis, vacuolation, breakdown of the cell wall, and hyphal death. Hyphae exposed to New Zealand isolates of E. aerogenes exhibited vacuolation of the outer cytoplasm and degradation of the cell wall and plasmalemma but not cell degradation and death.

Characteristics of Alternaria alternata Strawberry Pathotype Isolated in New Zealand and the Resistance of the 'Akita Berry' Strawberry to the Fungus

Cultural characteristics, morphology, and pathogenicities of ICMP-2487, a New Zea-land isolate of Alternaria alternata strawberry pathotype were compared with 5 Japanese isolates (OH-5, 90-12, MO-1, SN-3, and MA-2). Concurrently, the resistance of a new strawberry cultivar designated as 'Akita Berry', a somaclonal variant to a New Zealand isolate (ICMP-2487) was examined. The following results were obtained.1. No differences between the New Zealand and Japanese isolates were found in the growth and colors of colonies, shapes, sizes, and septa of conidia.2. Pathogenicities of ICMP-2487 to 58 strawberry cultivars and strains, 4 pear culti-vars, 3 cabbage cultivars, 3 tomato cultivars, 3 Welsh onion cultivars, 2 carrot cultivars, and 2 sunflower cultivars were similar to those of Japanese isolates.3. No lesions on 'Akita Berry' were formed by inoculation of ICMP-2487 and Japanese isolates. This cultivar is, therefore, resistant to the isolates of Alternaria alternata straw-berry pathotype occuring in Japan and in New Zealand.

Geographic variation of Chondrostereum purpureum detected by polymorphisms in the ribosomal DNA

Variation in the ribosomal (rDNA) repeat was analyzed for 107 isolates of the pathogenic fungus Chondrostereum purpureum, collected from Europe, New Zealand, and North America. The rDNA repeat of a representative Canadian isolate of C. purpureum was cloned into the λ vector EMBL-3, and a detailed restriction map was constructed. Variation in the large non-transcribed spacer region of the rDNA was determined for the entire collection of isolates following amplification by the polymerase chain reaction (PCR) and analysis of restriction fragment length polymorphisms (RFLPs). Three distinct nuclear type patterns were identified using the restriction endonuclease HaeIII. Nuclear type I was found in North American, European, and New Zealand isolates. Nuclear type II was only detected in isolates collected from North America, and nuclear type III was observed in isolates collected from both Europe and New Zealand. Nuclear type I was the predominant nuclear type in eastern North America as indicated by a frequency of 0.78, and nuclear type II occurred with a frequency of 0.89 in western North America. Gene flow across the continent was indicated by nearly equal nuclear type distributions (nuclear type I, 0.41; nuclear type II, 0.59) in central North America, but geographic separation has led to unequal nuclear-type distributions across North America. Keywords: Chondrostereum purpureum, biological control, genetic variation, ribosomal DNA.

Restriction fragment length polymorphisms in Australian and New Zealand isolates of Leptospira interrogans serovar pomona

Restriction fragment length polymorphisms in Australian and New Zealand isolates of <i>Leptospira interrogans</i> serovar pomona

Characterization of a temperate phage isolated fromLeuconostoc oenos strain 1002

A temperate phage was induced by mitomycin C fromLeuconostoc oenos strain 1002, a New Zealand isolate. The phage has an isometric head (52 ± 3 nm) with a tail (210 ± 10 nm) and gives a lytic burst size of 25 when grown on a sensitive strain. Three major structural proteins of 43, 30 and 14 kDa are visible by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). A restriction map of the 36-kb genome was determined. Commercially availableL. oenos strains PSU-1, ML34 and Lco23 and 46.6% of New Zealand isolates were sensitive to the phage.

Morphology and growth characteristics ofChrysochromulinaspecies (Haptophyceae = Prymnesiophyceae) isolated from New Zealand coastal waters

Chrysochromulina acantha, C. hirta, C. pachycylindra , and C. cf. polylepis (Hapto- phyceae = Prymnesiophyce ae) are new records, based on transmission electron microscopy, for New Zealand coastal waters. The morphology and growth characteristics of C. acantha and C. hirta, and of C. simplex and C. ericina (also New Zealand isolates), are described. C. ericina of the present study has a shorter haptonema (12-24 urn; non- coiling), larger plate and spine scales, and fewer spine scales (with broader bases), than northern hemisphere isolates. Scales of C. simplex fit the emended type description, whereas the large spine scales of C. hirta are shorter (< 11 um) than the type description (20—30 um). C. ericina and C. hirta fall into a temperate (15—20°C), and C. acantha and C. simplex into a sub-tropical (20—25°C), temperature group. All species tested grew well in brackish salinities (c. 23 through to 36); C. hirta grew optimally at a salinity of 23. All tolerated 25— 150 umol nr 2 s1 photon flux and pH of 7.5 to c. 8.4. C. acantha had significantly faster growth rates (doublings d~') with nitrate than with urea or ammonium chloride as nitrogen source, and C. hirta with urea and ammonium chloride rather than with nitrate; none required selenium. None of the species tested was toxic to Artemia salina. C. ericina and C. acantha were phagotrophic. Calcofluor assisted in the identification of C. quadrikonta from other spine-bearing species and FITC-conjugated wheat germ lectin aided differentiation of the Chryso- chromulina species from Prymnesium parvum and P. patelliferum.

Multilocus enzyme electrophoresis typing of New Zealand Listeria monocytogenes isolates

Eighty-five strains of Listeria monocytogenes, comprising 74 strains isolated from the environment, humans, animals and foods in New Zealand; 10 strains from similar sources in Denmark, and one reference strain, were studied using multilocus enzyme electrophoresis (MEE). Fifty-five electrophoretic types (ETs) were identified among the 85 strains, most (71%) of which were represented by only one isolate. The 74 New Zealand isolates produced 45 ETs, most (67%) of which were represented by only one isolate. There was no link between the source of the isolates and ET, as most ETs contained only one isolate. One cluster of ETs, designated cluster 45, contained only environmental isolates. Of the five ETs that contained isolates from more than one source, four contained isolates that were obtained from clinical specimens and foods indicating a possible link between isolates from these two sources. The population of New Zealand isolates of L. monocytogenes studied appears to be genetically diverse when compared with the diversity found in similar studies in other countries.

FITC‐conjugated lectins as a tool for differentiating between toxic and non‐toxic marine dinoflagellates

Abstract FITC‐conjugated lectins proved to be effective probes for differentiating between morphologically similar dinoflagellate species isolated from New Zealand coastal waters. In particular the binding (fluorescence) of peanut (PNA) lectin differentiated G. mikimotoi from Gymnodinium sp. (Waimangu) and G. pulchellum and the non‐binding of Helix pomatia (HPA) and wheat germ (WGA) lectins discriminated between G. mikimotoi and the other Gymnodinium species tested. G. breve (Florida) was differentiated from the New Zealand isolates by binding with soy bean (SBA) lectin. Ulex europeus (UEA) distinguished the toxic species Alexandrium minutum from the morphologically similar, but non‐toxic, Cachonina hallii. Two strains of Prorocentrum lima (Spain and Rangaunu) were not differentiated by the lectins, but P. lima was differentiated from P. compressum.

Comparison of Pulsed-Field Gel Electrophoresis DNA Fingerprints of Field Isolates of the Entomopathogen Bacillus popilliae

Eight strains of the entomopathogen Bacillus popilliae were analyzed by pulsed-field gel electrophoresis, and genomic size estimates of ;sim2,600 to 3,500 kb were obtained. The type strain, ATCC 14706, had a genomic size of 3,395 kb. For the six New Zealand isolates, the degree of similarity in the pulsed-field gel electrophoresis fingerprints may correlate with the geographical closeness of the sites of isolation. The plasmid profiles of the New Zealand isolates were also compared; four of the six strains carry plasmids in the 3.6- to 9.7-kb size range.

Siderophore production by New Zealand strains ofVibrio anguillarum

Abstract New Zealand isolates of Vibrio anguillarum from water, sediments and healthy salmon were tested for their ability to grow under iron‐limiting conditions and to produce sidero‐phores. Their growth in the presence of the iron chelator ethylenediamine‐di(o‐hydroxyphenyl‐acetic acid) (EDDA) suggested they could withstand conditions of iron limitation, with a 200 μM EDDA MIC recorded for the majority of strains. A positive reaction in the chrome azurol sulphate assay after growth in a low iron‐containing medium, which was negated when the medium was supplemented with 10 μM iron, indicated they could produce siderophores. The siderophores were of the phenolate class and their biological activities indicated they were related to enterobactin.

Phylogeny of Metarhizium: analysis of ribosomal DNA sequence data

Parsimony and neighbour joining analysis of complete sequence data of the internal transcribed spacer regions and the 5.8 s gene of 19 unique sequences from 31 fungal isolates supports Metarhizium as a monophyletic group. Three species were recognized: M. anisopliae, M. flavoviride and M. album. M. anisopliae and M. flavoviride/M. album represent two separate evolutionary lines within Metarhizium. Within M. anisopliae two groups are recognized, informally designated group A and B of M. anisopliae. It is likely that one of these corresponds to M. anisopliae var. anisopliae and the other represents a new variety of M. anisopliae which requires formal description. Two isolates within group A of M. anisopliae correspond to M. anisopliae var. majus. Three New Zealand isolates (FI698, FI699 and FI702) originally assigned to M. anisopliae fall into the M. flavoviride and M. album group; their phylogenetic position warrants further investigation. Parsimony analysis, in combination with T-PTP testing, demonstrates that rDNA sequence data can be used to resolve evolutionary relationships within Metarhizium.

Comparison of growth characteristics of New Zealand isolates of the prymnesiophytes Chrysochromulina quadrikonta and C.camella with those of the ichthyotoxic species C.polylepis

Chrysochromulina quadrikonta (Prymnesiophyceae), a quadriflagellate species previously unrecorded in New Zealand, was isolated from Nelson Harbour, New Zealand, in autumn 1991. It bears unmineralized plate and spine scales, which morphologically are most like those of Chrysochromulina ericina . Chrysochromulina quadrikonta, Chrysochromulina camella (isolated from the Marlborough Sounds, New Zealand) and Chrysochromulina polylepis (an ichthyotoxic species originally isolated from Scandinavia) grew most rapidly (growth rates, or divisions per day, of 1.41, 1.49 and 1.43, respectively) when cultured in a seawater-based general-purpose nutrient medium at a salinity of 24% and pH 7.9, with only C.camella still growing at 42% Chrysochromulina quadrikonta and C.camella grew optimally at 25°C. and C.polylepis between 15 and 20°C; only C.polylepis grew at 10°C. Chrysochromulina quadrikonta grew optimally with potassium nitrate and ammonium chloride as nitrogen source, whereas C.camella and C.polylepis grew equally well with urea as nitrogen source. Only C.quadrikonta and C.polylepis had a selenium requirement. Unlike C.polylepis , neither of the New Zealand isolates was phagotrophic nor ichthyotoxic.

Antibiotic susceptibilities, serotypes and auxotypes of Neisseria gonorrhoeae isolated in New Zealand.

The aim of the study was to determine the distribution of auxotypes and serotypes and the prevalence of antibiotic resistance among New Zealand isolates of Neisseria gonorrhoeae. A total of 486 gonococci isolated in 1988 were auxotyped, serotyped, and tested for susceptibilities to ten antibiotics. The gonococci were susceptible to all the antibiotics tested except penicillin and tetracycline. Eleven (2.2%) produced beta-lactamase, one (0.2%) showed chromosomal penicillin resistance, and 18 (3.7%) were resistant to a low-level of tetracycline. Most of the gonococci belonged to six auxotypes. The three predominant auxotypes were arginine-requiring (Arg-), non-requiring (NR), and arginine, hypoxanthine, uracil-requiring (AHU-). The majority of the isolates belonged to serogroup IB and to six serovars. The most prevalent serovars were IB-3 and IB-1. There was an association between penicillin susceptibility and auxotype or serovar among non-penicillinase producing N. gonorrhoeae (PPNG) isolates. Antibiotic resistance, including penicillin resistance, remains uncommon among gonococci in New Zealand. Baselines have been established for future epidemiological studies using both auxotyping and serotyping.

Characterisation of avian adenoviruses associated with inclusion body hepatitis

Eleven avian adenoviruses were isolated in monolayer cultures of specific pathogen free chicken kidney cells which were inoculated with suspensions of liver, intestine or bursa obtained from 15 broiler flocks experiencing outbreaks of inclusion body hepatitis (10 isolates) and from five unaffected flocks (one isolate). Of the 11 isolates obtained, nine were identified by virus neutralisation tests as serotype 8, one as serotype 1 and one as serotype 12. Adeno-associated viruses were only observed in combination with adenoviral particles of the serotype 12 isolate which was derived from a relatively mild outbreak of inclusion body hepatitis. Only the serotype 1 isolate, obtained from the unaffected broiler flock, consistently caused the death of embryos with marked pathological changes. All of the isolates produced basophilic intranuclear inclusion bodies surrounded by clear halos in chicken kidney cell cultures. DNA preparations, obtained from six strains of serotype 8 avian adenovirus (two New Zealand isolates, three Australian isolates and the reference strain HVI) after digestion with the restriction enzymes EcoRI and BamHI, gave electrophoretic patterns showing the New Zealand isolates to be similar to one another and to strain HVI, but quite distinct from the Australian isolates.

Genetic diversity among Australian and New Zealand isolates of Leptospira interrogans serovar pomona.

Restriction endonuclease analysis of 16 Australian and 4 New Zealand isolates of Leptospira interrogans serovar pomona showed that they could be divided into 3 genetic groups. Most of the isolates closely resembled the serovar kennewicki reference strain, and they all differed from the reference strain of serovar pomona. Based on these findings, it is suggested that vaccine manufacturers re-evaluate their choice of serovar pomona vaccine strain.

Neutralizing epitopes of feline calicivirus

A new collection of eighteen neutralizing monoclonal antibodies (N-MoAbs), raised against feline calicivirus (FCV), was used to analyze neutralizing epitopes of the F4 strain of FCV, the prototype strain of FCV in Japan. By cross-neutralization tests with the 20 FCV strains including Japanese. American, Swiss, and New Zealand isolates, the 18 N-MoAbs were categorized into six groups. One N-MoAb (1 D 7) neutralized all the strains tested: eight N-MoAbs neutralized only FCV-F 4; while the others neutralized the FCV strains in various degrees. In order to confirm this grouping, eight N-MoAbs were used to select neutralization-resistant variants of FCV F4. Although no variant against 1 D 7 was obtained, antigenic variants against other N-MoAbs were obtained. Neutralization tests using these variants revealed that there are six neutralizing epitopes on FCV F4 and that several epitopes are functionally related. One of these epitopes was the same epitope as one of the two epitopes identified by another panel of N-MoAbs we produced previously. Therefore, a total of seven neutralizing epitopes on FCV F4 were identified. Immunoblot analysis indicated that four of the seven epitopes existed on the 67 kDa capsid protein of the virus.

Characterisation by restriction endonuclease analysis and plasmid profiling ofVibrio ordaliistrains from salmon(oncorhynchus tshawytschaandoncorhynchus nerka)with vibriosis in new zealand

Abstract Isolates of Vibrio ordalii were recovered from salmon (Oncorhynchus tshawytscha and Oncorhynchus nerka) cultured in New Zealand. The fish suffered from clinical vibriosis, from seven outbreaks in five geographically distinct areas. Affected fish showed gross signs and histopathology similar to those reported from North America. The presence of a plasmid, designated pVO1, approximately 30 kilobases (kb) in size, was seen in all V. ordalii strains examined This plasmid was identical in size and restriction endonuclease cleavage analysis to a plasmid (pMJ101) previously found in V. ordalii strains elsewhere. After togging pVO1 with the transposon Tn1 from the R plasmid RP4, attempts to isolate plasmid‐less derivatives using a variety of curing agents were unsuccessful. All of the New Zealand isolates possessed identical chromosomal DNA cleavage patterns. These patterns were only slightly different from that of the type strain but substantially different from that of the Vibrio anguillarum type strain.