Zealand Isolates Research Articles

Fungal Inoculum Properties: Extracellular Enzyme Expression and Pentachlorophenol Removal in Highly Contaminated Field Soils

This study was conducted to improve the pentachlorophenol (PCP) bioremediation ability of white-rot fungi in highly contaminated field soils by manipulating bioaugmentation variables. These were the dry weight percentage of fungal inoculum addition (31-175 g kg(-1)), PCP concentration (100-2137 mg kg(-1) PCP), fungal inoculum formulation, and time (1-7 wk). Five fungal isolates were used: the New Zealand isolates Trametes versicolor (L.: Fr.) HR131 and Trametes sp. HR577; the North American isolates Phanerochaete chrysosporium Burds. (two isolates) and Phanerochaete sordida (Karst.) Erikss. & Ryv. Pentachlorophenol removal, manganese peroxidase, and laccase activity, and the formation of chloroanisoles in the contaminated field soils were measured. The majority of PCP removed by the Trametes isolates was in the first week after bioaugmentation. The maximum PCP removal by the fungi varied from 50 to 65% from a 1065 mg kg(-1) PCP contaminated field soil. Pentachlorophenol was preferentially converted to pentachloroanisole (PCA) by the Phanerochaete isolates (>60%), while 2 to 9% of the PCP removed by two Trametes isolates was converted to PCA. A pH increase was measured following bioaugmentation that was dependent on PCP concentration, fungal inoculum addition, and formulation. This, together with rapid initial PCP removal, possibly changed the bioavailability of the remaining PCP to the fungi and significantly decreased the sequestering of PCP in the contaminated field soils. The research supports the conclusion that New Zealand Trametes spp. can rapidly remove PCP in contaminated field soils. Bioavailability and extractability of PCP in the contaminated field soil may significantly increase after bioaugmentation.

Identification of Five Major and Two Minor Genotypes of Varicella-Zoster Virus Strains: a Practical Two-Amplicon Approach Used To Genotype Clinical Isolates in Australia and New Zealand

Whole genome phylogenetic analysis in this study resolved a total of five major genotypes among the 22 varicella-zoster virus (VZV) strains or isolates for which complete genomic sequences are available. Consistent with earlier publications we have designated these genotypes European 1 (E1), European 2 (E2), Japanese (J), mosaic 1 (M1), and mosaic 2 (M2). Single nucleotide polymorphism (SNP) analysis performed in a whole-genome alignment revealed that VZV isolates of all five genotypes can be accurately genotyped using SNPs from two amplicons: open reading frame 22 (ORF22) and either ORF21 or ORF50. This modified approach identifies all of the genotypes observed using any of the published genotyping protocols. Of 165 clinical varicella and zoster isolates from Australia and New Zealand typed using this approach, 67 of 127 eastern Australian isolates were E1, 30 were E2, 16 were J, 10 were M1, and 4 were M2; 25 of 38 New Zealand isolates were E1, 8 were E2, and 5 were M1. VZV strain diversity in eastern Australia is thus broader than has been described for any other region, including Europe, Africa, and North America. J strains were far more prevalent than previously observed in countries other than Japan. Two-amplicon typing was in complete accord with genotypes derived using SNP in multiple ORFs (ORFs 1, 21, 22, 38, 50, 54, and 62). Two additional minor genotypes, M3 and M4, could also be resolved using two-amplicon typing.

Characterisation and bioactivity of protein-bound polysaccharides from submerged-culture fermentation of Coriolus versicolor Wr-74 and ATCC-20545 strains

The protein-bound polysaccharides of Coriolus versicolor (CPS) have been reported to stimulate overall immune functions against cancers and various infectious diseases by activating specific cell functions. A New Zealand isolate (Wr-74) and a patented strain (ATCC-20545) of C. versicolor were compared in this study. The fruit bodies of both strains were grown for visual verification. Both strains were grown in submerged-culture using an airlift fermentor with milk permeate as the base medium supplemented with glucose, yeast extract and salt. Metabolic profiles of both strains obtained over 7-day fermentation showed very similar trends in terms of biomass production (8.9-10.6 mg/ml), amounts of extracellular polysaccharide (EPS) from the culture medium (1150-1132 microg/ml), and intracellular polysaccharide (IPS) from the mycelium (80-100 microg/ml). Glucose was the dominant sugar in both EPS and IPS, and the polymers each consisted of three molecular weight fractions ranging from 2 x 10(6) to 3 x 10(3 )Da. Both the EPS and IPS were able to significantly induce cytokine production (interleukin 12 and gamma interferon) in murine splenocytes in vitro. Highest levels of interleukin 12 (291 pg/ml) and gamma interferon (6,159 pg/ml) were obtained from samples containing Wr-74 IPS (0.06 microg/ml) and ATCC 20545 IPS (0.1 microg/ml), respectively. The results indicated that lower levels of EPS and IPS generally resulted in higher immune responses than did higher polymer concentrations.

Distinct patterns of evolution between respiratory syncytial virus subgroups A and B From New Zealand isolates collected over thirty‐seven years

Respiratory syncytial virus (RSV) is the most important cause of viral lower respiratory tract infections in infants and children worldwide. In New Zealand, infants with RSV disease are hospitalized at a higher rate than other industrialized countries, without a proportionate increase in known risk factors. The molecular epidemiology of RSV in New Zealand has never been described. Therefore, we analyzed viral attachment glycoprotein (G) gene sequences from 106 RSV subgroup A isolates collected in New Zealand between 1967 and 2003, and 38 subgroup B viruses collected between 1984 and 2004. Subgroup A and B sequences were aligned separately, and compared to sequences of viruses isolated from other countries during a similar period. Genotyping and clustering analyses showed RSV in New Zealand is similar and temporally related to viruses found in other countries. By quantifying temporal clustering, we found subgroup B viruses clustered more strongly than subgroup A viruses. RSV B sequences displayed more variability in stop codon usage and predicted protein length, and had a higher degree of predicted O-glycosylation site changes than RSV A. The mutation rate calculated for the RSV B G gene was significantly higher than for RSV A. Together, these data reveal that RSV subgroups exhibit different patterns of evolution, with subgroup B viruses evolving faster than A.

Phylogenetic Analysis of “Candidatus Phytoplasma australiense” Reveals Distinct Populations in New Zealand

ABSTRACT The phytoplasma "Candidatus Phytoplasma australiense" has been reported from New Zealand and Australia, where it has been associated with a range of host plants, especially since the 1970s. Partial tuf gene sequences of 36 New Zealand (NZ) isolates from four different host genera revealed nine different variants, which clustered into two distinct groups without any obvious correlation with host or geographic region. Phylogenetic analysis of these sequences, together with those available from Australian isolates, revealed three distinct clades: one found solely in Australia, one found solely in NZ, and a third with representatives from both countries. These divisions are consistent with differences observed in the 16-23S rRNA internal transcribed spacer region; therefore, we conclude that they represent three distinct subgroups: tuf 1, tuf 2, and tuf 3. We estimated a time of divergence for the three clades based on a synonymous substitution rate calculated by comparing the complete tuf gene sequence from the Loofah witches'-broom phytoplasma and "Candidatus Phytoplasma australiense". Using a calibration date of 110 million years, the estimated time to a common ancestor for all clades (6 to 9 million years ago) suggests divergence during the Miocene, well after the geological separation of NZ and Australia.

Molecular characterisation of an Australian isolate of Bonamia exitiosa

An Australian (New South Wales) isolate of Bonamia was characterised at the molecular level by sequencing the 18S-ITS-1 region of the small subunit rRNA operon obtained from flat oysters Ostrea angasi shown to be infected by histological examination. Sequence data alignment with homologous genes from 2 other isolates of Bonamia (New Zealand and France) revealed high levels of nucleotide identity with both isolates. However, the Australian Bonamia is shown to be more closely related to the New Zealand isolate, suggesting the existence of an oceanic subgroup of Bonamia.

Application of Streptococcus uberis Multilocus Sequence Typing: Analysis of the Population Structure Detected among Environmental and Bovine Isolates from New Zealand and the United Kingdom

We recently developed a multilocus sequence typing (MLST) scheme to differentiate S. uberis isolates and facilitate an understanding of the population biology of this pathogen. The scheme was initially used to study a collection of 160 bovine milk isolates from the United Kingdom and showed that the majority of isolates were from one clonal complex (designated the ST-5 complex). Here we describe the MLST analysis of a collection of New Zealand isolates. These were obtained from diverse sources, including bovine milk, other bovine anatomical sites, and environmental sources. The complete allelic profiles of 253 isolates were determined. The collection was highly diverse and included 131 different sequence types (STs). The New Zealand and United Kingdom populations were distinct, since none of the 131 STs were represented within the previously studied collection of 160 United Kingdom S. uberis isolates. However, seven of the STs were members of the ST-5 clonal complex, the major complex within the United Kingdom collection. Two new clonal complexes were identified: ST-143 and ST-86. All three major complexes were isolated from milk, other bovine sites, and the environment. Carriage of the hasA gene, which is necessary for capsule formation, correlated with clonal complex and isolation from clinical cases of mastitis.

Development of an isolate-specific marker for tracking Phaeomoniella chlamydospora infection in grapevines

Petri disease causes decline of grapevines worldwide. The grapevine endophyte Phaeomoniella chlamydospora is the most important fungal pathogen associated with this disease. Epidemiological studies of this pathogen have been hampered by its common occurrence in the internal tissue of apparently healthy vines. Development of a molecular marker for a single strain would overcome this limitation and aid experiments designed to answer key questions about the biology of this pathogen. Genetic variation analysis of New Zealand and Italian strains of P. chlamydospora detected a potential molecular marker in New Zealand isolate A21. Characterization of the 1010 bp marker band showed that it had 50% identity to moxY, a gene involved in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. Sequencing of the region flanking the 1010 bp product revealed a single nucleotide polymorphism in the 3′ border of the marker band. Primers were designed to amplify a 488 bp fragment encompassing this polymorphic site and cleavage of this product with the restriction enzyme BsrI produced three bands only in isolate A21 and two bands in all other isolates tested. The sensitivity of the PCR-RFLP protocol was increased with a nested PCR approach and the protocol optimized for soil and wood samples. When the nested PCR/RFLP procedure was used to determine the persistence of viable and nonviable spores in soil, the results showed that nonviable spores were undetected after 8 wk whereas viable spores still could be detected at 17 wk.

Development of an isolate-specific marker for tracking Phaeomoniella chlamydospora infection in grapevines

Petri disease causes decline of grapevines worldwide. The grapevine endophyte Phaeomoniella chlamydospora is the most important fungal pathogen associated with this disease. Epidemiological studies of this pathogen have been hampered by its common occurrence in the internal tissue of apparently healthy vines. Development of a molecular marker for a single strain would overcome this limitation and aid experiments designed to answer key questions about the biology of this pathogen. Genetic variation analysis of New Zealand and Italian strains of P. chlamydospora detected a potential molecular marker in New Zealand isolate A21. Characterization of the 1010 bp marker band showed that it had 50% identity to moxY, a gene involved in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. Sequencing of the region flanking the 1010 bp product revealed a single nucleotide polymorphism in the 3' border of the marker band. Primers were designed to amplify a 488 bp fragment encompassing this polymorphic site and cleavage of this product with the restriction enzyme BsrI produced three bands only in isolate A21 and two bands in all other isolates tested. The sensitivity of the PCR-RFLP protocol was increased with a nested PCR approach and the protocol optimized for soil and wood samples. When the nested PCR/RFLP procedure was used to determine the persistence of viable and nonviable spores in soil, the results showed that nonviable spores were undetected after 8 wk whereas viable spores still could be detected at 17 wk.

Characterization of major clones of antibiotic-resistant Streptococcus pneumoniae in New Zealand by multilocus sequence typing

To determine the relationship between New Zealand isolates of antibiotic-resistant Streptococcus pneumoniae and internationally widespread resistant clones. Fifty-nine isolates representing both invasive and non-invasive pneumococci from multiple locations, serotypes and years were analysed by multilocus sequence typing. Major international clones, including Spain(23F)-1, France(9V)-3 and Taiwan(19F)-14, were found to be present in New Zealand. A one-allele variant of the Taiwan(19F)-14 clone (aroE 15-->4, ST 271) was particularly prominent. Antibiotic-resistant pneumococci have not evolved de novo in New Zealand, but were introduced to the country during the early 1990s.

British‐based search for natural enemies of the clover root weevil, Sitona lepidus in Europe

Since the discovery of the clover pest Sitona lepidus (Coleoptera: Curculionidae) in New Zealand in 1996, substantial effort has been made to find suitable biological control agents. In the summer of 2000 about 8600 Sitona lepidus were collected from 15 locations in 11 European countries. The most common parasitoid recovered was Microctonus aethiopoides (Hymenoptera: Braconidae). These parasitoids were used to infect S. lepidus populations that were then forwarded to a quarantine facility in New Zealand. All European‐derived populations of this parasitoid were found to develop readily in both British and New Zealand populations of S. lepidus. This contrasted with the New Zealand ecotype of M. aethiopoides which was unable to develop in S. lepidus. The S. lepidus population obtained from Caen, France was also parasitised by a Perilitus sp. (Hymenoptera: Braconidae) along with Microsoma exiguum (Diptera: Tachinidae). Microsoma exiguum was also reared from weevils collected in Devon along with a single female specimen of Allurus litu‐ratus (Hymenoptera: Braconidae). Of all the species reared out however, only M. aethiopoides could be induced to parasitise S. lepidus in the laboratory. Thirty‐four different isolates of Beauveria bassiana (Balsamo) Vuillemin (Deuteromycotina: Hyphomy‐cetes) strains were collected, some of which were more virulent than New Zealand isolates already tested against adult S. lepidus. Fifteen isolates of the facultative pathogen Serratia Bizio spp. (Enterobac‐teriaceae) were also collected.

Two novel cytotoxic cyclodepsipeptides from a mycoparasitic Cladobotryum sp.

Two novel cyclodepsipeptides (1 and 2) along with three known compounds (3-5) were isolated from a New Zealand isolate of a Cladobotryum sp. The structures of 1 and 2 were elucidated from NMR and HRESIMS experiments, and the absolute stereochemistry of 2 was established by X-ray crystallography and chemical degradation. Compounds 1 and 2 were both cytotoxic against murine P388 leukemia cells.

Low genetic variation detected in New Zealand populations of Phaeomoniella chlamydospora

Phaeomoniella chlamydospora is considered to be the causal agent of Petri disease. This disease causes decline of grapevines in most grape growing regions of the world. Genetic variation within 39 New Zealand isolates of Phaeomoniella chlamydospora was compared to six isolates from Italy using randomly amplified polymorphic DNA (RAPD), randomly amplified microsatellites (RAM), amplified fragment length polymorphism (AFLP), and universally primed polymerase chain reaction (UP-PCR). Using each method, genetic variation within New Zealand and Italian isolates of P. chlamydospora was shown to be low, with a maximum of seven genetic groups identified by each primer. The greatest amount of genetic variation was shown using AFLP analysis, with 21 different groups identified. RAPD, AFLP and UP-PCR primers detected inter-vineyard, intra-vineyard and intra-vine variation of New Zealand isolates. A subset of five New Zealand and one Italian isolate was further investigated using mycelial compatibility groups (MCGs). One MCG was identified, supporting low genetic variation within P. chlamydospora isolates. Low genetic variation within the New Zealand and the Italian populations suggests that asexual reproduction predominates, and the presence of intra-vineyard and intra-vine variation in New Zealand indicates that multiple introductions have occurred.

Occurrence, morphological characteristics and ribotyping of New Zealand isolates of Duddingtonia flagrans, a candidate for biocontrol of animal parasitic nematodes

The nematophagous fungus Duddingtonia flagrans has shown potential for biological control of nematode parasites of livestock in several countries. A survey for the presence of D. flagrans in New Zealand was undertaken in 1997–98 on 24 farm, research, and domestic properties in the southern North Island. The nematophagous fungi found on isolation plates included species of Arthrobotrys, Dactylellina, Nematoctonus, Harposporium, and Duddingtonia. Isolates that produced the three‐dimensional nets and abundant chlamydospores, characteristic of D. flagrans, were detected in 6 of 205 samples examined. The fungus was obtained from a variety of substrates such as cattle dung, horse dung, seepage from a silage pit, and decomposing lawn clippings. Morphological characteristics, and the sequences of ITS/5.8 region of ribosomal DNA, were very similar to those published for D. flagrans. The fungus was re‐isolated from the faeces of lambs 16–40 h after they had been dosed orally with chlamydospores, indicating survival of gut passage.

The Complete Nucleotide Sequence of a Japanese Isolate of White clover mosaic virus Strain RC

The complete nucleotide sequence was determined for genomic RNA of White clover mosaic virus (WClMV-RC) isolated from red clover (Trifolium pratense) in Japan, It is 5843 nucleotides in length, excluding the poly(A) tail at the 3' terminus. Similar to other potexviruses, it contains five open reading frames (ORFs 1 through 5), which putatively encode an RNA-dependent RNA polymerase (RdRp) (147 kDa), a triple gene block (TGB) (26 kDa/13 kDa/7 kDa), and a coat protein (CP) (22 kDa), respectively. The deduced amino acid sequence of the WClMV-RC CP was identical to that of WClMV-O, one of two New Zealand isolates, but only 85% identical to that of WClMV-M, the other New Zealand isolate, because of heterogeneity in the C-termini of CP amino acid sequences. The implication of this CP heterogeneity is discussed.

Variation among New Zealand isolates of Sphaeropsis sapinea

Sphaeropsis sapinea is a pathogen of many coniferous species and causes significant losses to the plantation forestry industries of many countries. New Zealand isolates of S. sapinea were examined for colony and morphological characteristics, and pathogenicity to Pinus radiata seedlings. Considerable variation was observed in colony growth form and colour, and in the growth rates on four agar media. The conidial dimensions, carbon and nitrogen utilization, and pathogenicity also varied among the isolates. The variation observed was such that the isolates could not be placed within previously described distinct morphotypes and the present results concur with previous studies cautioning the use of morphological criteria alone to partition the species. These results contrast markedly with previous reports and seemingly indicate a change in the New Zealand S. sapinea population. This difference may reflect the re‐introduction or spread of additional isolates or a shift in the structure of the resident population.

Genetic diversity in New Zealand populations of Sclerotium cepivorum

Mycelial interactions were examined between 231 New Zealand isolates of Sclerotium cepivorum Berk., as well as 25 isolates from a further eight countries. Within the group of New Zealand isolates six mycelial compatibility groups (MCGs) were identified and named using reference strains obtained from the University of Guelph, Canada. Two new groups, presently only known from New Zealand, were identified. Heterogeneity was found both within and between fields in all regions. A subset of 51 S. cepivorum isolates was further investigated for genetic diversity using universally primed PCR (UP‐PCR) and random amplified polymorphic DNA (RAPD) analyses. When results were analysed separately, each primer distinguished one to three genetic groups. However, when the results from two primers (L15 and OPAX15) were combined, six genetic groups were revealed. The results, as shown by MCGs and molecular methods, demonstrate the presence of genetic diversity in New Zealand field populations of S. cepivorum.

Soil on imported shipping containers provides a source of new Pseudomonad biodiversity into New Zealand

Exotic soil attached to shipping containers imported to New Zealand was assessed for new species of Pseudomonas (sensu stricto) bacteria to determine whether this soil facilitated the introduction of new biodiversity. Pseudomonads were isolated from exotic soil originating from 19 different countries (termed container isolates (CIs)), and from soils in eight geographically distinct locations throughout New Zealand (termed New Zealand isolates (NZIs)). Partial 16S rRNA gene nucleotide sequences were determined from 40 selected CIs and 38 NZIs. Relationships between CIs, NZIs, and a further 25 typed Pseudomonad species were inferred by 16S rRNA phylogenetic analyses. CIs showed relatedness to a broad range of Pseudomonad species and many CIs grouped with NZIs suggesting they are closely related to Pseudomonads currently in New Zealand. However, unique phylogenetic branchings of CIs also indicate that soils attached to shipping containers may provide a source of new Pseudomonad biodiversity to New Zealand.

Molecular evidence for the presence of Phaeomoniella chlamydospora in New Zealand nurseries, and its detection in rootstock mothervines using species-specific PCR

Phaeomoniella chlamydospora is considered to be the causal agent of Petri disease of grapevines. The source of primary inoculum is believed to be through the planting of grafted vines collected from infected mother rootstock vines. The presence of P. chlamydospora in New Zealand has been confirmed by analysis of the ribosomal gene region amplified from morphologically identified isolates. Sequencing of six of these isolates revealed that their nucleotide sequences were 99.9–100% similar to those reported in GenBank, and established that the species-specific primers, previously developed for overseas strains, would amplify the New Zealand isolates. A method for DNA extraction from grapevine wood was developed which, when combined with the species-specific primers, provides the basis for a diagnostic service for the detection of this disease in rootstock.

Impact of a North American isolate of the microsporidium Nosema carpocapsae on a laboratory population of the codling moth, Cydia pomonella.

Nosema carpocapsae is a microsporidian pathogen of the codling moth, Cydia pomonella. We report the occurrence of this pathogen in a colony originating from collections made in the United States. This is the first record of N. carpocapsae infecting North American codling moths. This North American isolate of N. carpocapsae was indistinguishable from isolates received from New Zealand and Bulgaria, based on small subunit ribosomal RNA sequencing, but was more virulent than the previously described New Zealand isolate. In the laboratory, infected larvae and pupae had increased mortality compared to their uninfected counterparts and developmental time increased by 1 week. There was no effect on female fecundity. Within a cohort of eggs laid by infected females, neonates that emerged first were more likely to be uninfected. We established an uninfected colony by interrupting horizontal transmission and only utilizing the larvae that emerged from the first-laid eggs.