Denis R. Lauren

Fusarium graminearum, F. cortaderiae and F. pseudograminearum in New Zealand: molecular phylogenetic analysis, mycotoxin chemotypes and co-existence of species

Fusarium graminearum and F. pseudograminearum are important plant pathogens in New Zealand and around the world. Headblight and crown rot diseases of cereals caused by these species are responsible for large economic losses due to reduction in seed quality and contamination of grain with tricothecene mycotoxins. In the current study we have used two different molecular phylogenetic approaches, AFLPs and gene genealogies, to gain insight into the evolutionary relationships between F. graminearum, and F. pseudograminearum in New Zealand. The worldwide genetic diversity of F. graminearum clade is represented by at least eight biogeographically distinct species (previously designated as lineages of F. graminearum). Our analysis demonstrated that this clade is represented by F. graminearum (= F. graminearum Lineage 7) and F. cortaderiae (= F. graminearum Lineage 8) in New Zealand. Through our analysis we also confirm the presence of F. pseudograminearum in New Zealand as a first record for this organism. Information on species is necessary for preventing the inadvertent intercontinental introduction of genetically unique foreign pathogens associated with world trade. The ability to place species information into a worldwide context enabled postulation that the New Zealand representatives of F. graminearum clade originated from at least two regions, and probably on at least two hosts. Correlation of species descriptions with biogeographical and host information revealed evidence for co-localisation of F. graminearum clade species with potential for genetic outcrossing in the field. Mycotoxin analysis showed F. graminearum (= lineage 7) isolates produce either nivalenol (NIV) or deoxnivalenol (DON). In contrast, F. cortaderiae isolates produced only NIV. These findings support earlier observations that mycotoxin production in the F. graminearum clade is not species specific, but suggest maintenance of chemotype diversity through speciation may have been restricted to a subset of species.

Isoptaquiloside and caudatoside, illudane-type sesquiterpene glucosides from Pteridium aquilinum var. caudatum

Ptaquiloside and two new illudane-type sesquiterpene glycosides were isolated from Pteridium aquilinum var. caudatum. One- and two-dimensional NMR analyses revealed the new glucosides to be isoptaquiloside and caudatoside. Four pterosins, A, B, K and Z, were also isolated from a base-acid treated extract.

Antifungal saponins from Paris polyphylla Smith.

Three steroidal saponins, including one new and two known compounds, were isolated from the rhizomes of Paris polyphylla Smith. One- and two-dimensional NMR, LC-MS, and interpretation of hydrolytic cleavage experiments led to the identification of the structure of the new saponin as ( 25R)-spirost-5-ene-3 beta,17 alpha-diol (pennogenin) 3- O-{ O- alpha- L-rhamnopyranosyl-(1-->2)- O-[ O- beta-xylopyranosyl-(1-->5)- alpha- L-arabinofuranosyl-(1-->4)]- beta- D-glucopyranoside}. The isolated saponins were evaluated for their antifungal activity against Cladosporium cladosporioides and Candida species and showed comparable activity to chemicals used in some commercial products.

Excretion, through milk, of ptaquiloside in bracken-fed cows. A quantitative assessment

The content of ptaquiloside (ptaq), a major Pteridium aquilinum carcinogen found in milk from cows fed bracken in Venezuela, has been estimated quantitatively in ils passage through bovines into milk. Six cows were given 6 kg/animal/d of fresh bracken fronds whose ptaq content was deter- mined. Ptaq first appeared in milk 38 h after initially feeding this plant to the cows and continued to be excreted at a relatively constant level, after an induction period, for 62-70 h after the bracken diet had ceased. The average value of total ptaq excreted ((ptaq)e) from milk amounted to 8.60 ± 1.16 % of the total ptaq ingested ((ptaq)) during the entire feeding period. (ptaq)e was linearly dose depen- dent in the 2 400 to 10 000 mg/animal/d range of ptaq given to the test animais and the ratio (ptaqle/jptaq), remained relatively constant at various ptaq dosages. In a two-pulse feeding sequence with a 72-h interlude when no bracken was given, a bimodal ptaq excretion curve was obtained. The implications to human health are discussed.

Mycotoxins in New Zealand maize: A study of some factors influencing contamination levels in grain

Abstract Samples of grain from New Zealand grown maize (Zea mays L.), collected from yield comparison strip trials at harvest during 1992, 1993, and 1994, were analysed for contamination by the Fusarium mycotoxins nivalenol (NIV), deoxynivalenol (DON), and zearalenone (ZEN). The samples collected in 1992 were also assessed for the incidence of infection by Fusarium spp. The samples mostly represented commercially grown hybrids, and 12 of the hybrids were sampled more extensively and were collected in each year of the study. The incidence of mycotoxin contamination was high in each year, especially by the two trichothecenes, NIV and DON. The levels found in 1992 were generally higher than in 1993 and 1994. Much of this was because of adverse weather conditions during the 1991/92 season which led to delayed harvest. In each year samples harvested after early June tended to have higher contamination levels. It was also observed in the 1992 season that Fusarium infection levels were higher in the later harves...

Trichoderma/pathogen interactions: measurement of antagonistic chemicals produced at the antagonist/pathogen interface using a tubular bioassay

A tubular bioassay was used to measure analytically the local production and concentration of the antifungal Trichoderma secondary metabolite 6‐n‐pentyl‐2H‐pyran‐2‐one (6PAP) at the Trichoderma antagonist/pathogen interface. 6PAP levels significantly increased in the presence of the pathogen Botrytis cinerea, typically 300–700%, and were highest near the pathogen source. The level of response for a particular Trichoderma isolate was found to vary with the test organism used. Two products produced by biotransformation of 6PAP by B. cinerea in response to the interaction were also detected.

In vitro effects of food extracts on selected probiotic and pathogenic bacteria

A panel of 148 extracts from 37 food products was prepared using organic and aqueous solvents and both neutral and acidic conditions. The panel of food products tested included fruits, vegetables, grains, herbs and spices, most of which are common in a normal European-style diet. The impact of these extracts on the growth of selected probiotic bacteria (Lactobacillus reuteri, Lactobacillus rhamnosus, Bifidobacteria lactis) and pathogenic bacteria (Escherichia coli 0157:H7 and Escherichia coli LF82) was assessed using a standard minimum inhibitory concentration method. The results showed that aqueous extractions of garlic and black peppercorns significantly enhanced the growth of one strain of probiotic bacteria (L. reuteri) whilst inhibiting both pathogenic strains of E. coli at a 1:50 dilution. Aqueous extracts of banana, apple and orange all enhanced the growth of the three probiotic strains significantly, and inhibited the pathogens to approximately 80% of the controls (not significant). Both aqueous and organic extractions of ginger significantly inhibited the growth of one or both E. coli strains, respectively (also at the 1:50 dilution).

Influence of harvest date and hybrid on the mycotoxin content of maize (Zea mays) grain grown in New Zealand

Abstract The changes in concentrations of the Fusarium mycotoxins nivalenol (NIV), deoxynivalenol (DON), and zearalenone (ZEN) in harvested maize (Zea mays) grain with time of harvest were determined, as well as how those changes were affected by the hybrid grown. The study used five hybrids in commercial use, three of these resistant to mycotoxin accumulation under New Zealand growth conditions (3902, 3787, 3394) and two less resistant (3751, 3514). The trial was conducted at five well‐maintained long‐term maize research sites over the three major maize‐growing regions of New Zealand. Results showed that apart from seasonal effects, hybrid choice was the major factor affecting the incidence and concentration of mycotoxins in harvested maize grain. Susceptible hybrids were significantly more likely to contain mycotoxins and to have high concentrations. It was determined that harvest date also had an impact on mycotoxin concentrations, with the best indicators of increased mycotoxin risk being either a prolonged time elapsed since the crop first reached 28% kernel moisture, or a very late (late June or July) harvest. The possibility of a site effect was also observed. At one site in the Waikato region, cob and kernel moisture contents were determined over most of the season, and the collected grain measured for infection by Fusarium species, and for contamination by mycotoxins. This showed that once the kernel moisture content fell below 30%, a substantial increase in Fusarium infection is likely. When this occurs, further delays in harvest can compromise grain quality and lead to increased mycotoxin contamination. The level of fungal infection and mycotoxin contamination would have been markedly less if the crops had been harvested earlier than they were, with the most susceptible hybrids being most affected. The study showed that although there may exist a reasonably wide harvest window for resistant hybrids planted at good sites, with more susceptible hybrids it is imperative to harvest as soon as practical, but that even then, mycotoxin contamination can be present.

Molecular imprinting of a small substituted phenol of biological importance

A polymer capable of recognising small phenolic molecules is described. Analysis based on batch binding experiments followed by HPLC was used to determine propofol concentration in different test solutions. Polymer particle size and contact time between propofol test solutions and polymers were modified to yield a fast and reliable system for propofol detection. The polymer thus synthesised had little cross-reactivity when tested against other structurally related phenols.

Variation in ptaquiloside content In bracken (Pteridium esculentum (Forst. f) Cockayne ) in New Zealand.

Abstract AIM: To examine stands of bracken fern (Pteridium esculentum) from throughout New Zealand for the presence and concentration of ptaquiloside (Pta), and to compare the presence and/or concentrations of Pta in areas where bovine enzootic haematuria (BEH) and/or acute haemorrhagic syndrome (AHS) has been known to occur with those where BEH/AHS has not been recorded. METHODS: Stands of bracken fern were sampled from 275 sites throughout New Zealand. Sixty-two stands were from a regional survey predominantly from the Waikato and Coromandel regions, 27 were from a farm in the King Country where BEH/AHS had been investigated previously, and 186 were from a national survey of the North and South Islands. Sampling sites were from a mixture of grazed paddocks, roadsides, and forest and bush areas. Samples comprised whole young fronds, the tops of unfurling young fronds, or, for the Regional Survey, mature green fronds from the previous season. Pta was extracted from the samples, and measured using high-performance liquid chromatography. Information on the occurrence of BEH/AHS at specific locations was obtained from published information and records from animal health laboratories in New Zealand. RESULTS: The 275 samples contained widely varying concentrations of Pta. In the Farm Survey, concentrations ranged from 280–13,300 (mean 3,800) µg/g (on a dry-weight basis) in the 63% of samples that contained Pta. A high proportion of samples from the Regional and National Surveys covering large areas of the country contained no detectable levels of Pta. The majority (61%) of samples from these two surveys which contained Pta were from areas where BEH/AHS was reported to occur. Combining data from all surveys, in areas with reported BEH/AHS, 42% of samples collected contained Pta, compared with 6% where BEH/AHS was not known to occur. CONCLUSIONS: Concentrations of Pta in bracken in New Zealand vary greatly, and in a high proportion of stands Pta is not found. A higher incidence of Pta, and some very high concentrations, are found in areas where BEH/AHS was known to occur.