Community Structure of Endophytic Actinobacteria in a New Zealand Native Medicinal Plant Pseudowintera colorata (Horopito) and Their Influence on Plant Growth.

The role of plant endophytic Actinobacteria remains poorly understood with no reports of these communities in New Zealand native plants. This first investigation of endophytic Actinobacteria in New Zealand targeted the culturally significant medicinal shrub Pseudowintera colorata (horopito) as a model plant. Community analysis in plant tissues collected from ten geographically distinct sites showed that tissue type had the strongest influence on diversity and richness of endophytic Actinobacteria. More denaturing gradient gel electrophoresis (DGGE) bands were obtained from stems (n = 18) compared to roots (n = 13). Sequencing analysis of the major bands (n = 20) identified them as uncultured bacteria, Streptomyces sp. and Angustibacter peucedani. Using two Actinobacteria-specific media, nine isolates were recovered from surface-sterilised P. colorata tissues. This was approximately 12% of the total taxa and correlated well with culturable numbers in international studies. In vitro analysis of the functionality of these strains showed that Streptomyces sp. PRY2RB2 inhibited all the tested phytopathogenic fungi (n = 4), Streptomyces sp. UKCW/B and Nocardia sp. TP1BA1B solubilised phosphate and produced siderophores. The functionality of the phosphate solubilising strains (n = 2) in vivo was investigated by inoculation of P. colorata seedlings. After 4 months, the mean shoot height of seedlings treated with Nocardia sp. TP1BA1B was 1.65× longer, had higher shoot dry weight (1.6×) and number of internodes (1.67×) compared to control. This study identified for the first time a key group of endophytic Actinobacteria that are likely to be important in the ecology of New Zealand flora.

Red leaf margins indicate increased polygodial content and function as visual signals to reduce herbivory in Pseudowintera colorata.

Red-pigmented leaf margins are common, but their functional significance is unknown. We hypothesized that red leaf margins reduce leaf herbivory by signalling to herbivorous insects the presence of increased chemical defences. Leaves were collected from a natural population of Pseudowintera colorata. Margin size, herbivory damage, anthocyanin content and concentrations of polygodial, a sesquiterpene dialdehyde with antifeedant properties, were quantified. Feeding trials involving larvae of Ctenopseustis obliquana, a generalist herbivore, were conducted on red- and green-margined P. colorata leaves in darkness, or under white, green or red light. Leaves with wider red margins contained higher concentrations of polygodial and anthocyanins, and incurred less natural herbivory. In trials under white light, C. obliquana consumed disproportionately more green- than red-margined leaf laminae. Larvae exhibited no feeding preference when light was manipulated such that leaf colour discrimination was impaired. Red leaf margins provide a reliable and effective visual signal of chemical defence in P. colorata. Ctenopseustis obliquana larvae perceive and respond to the colour of the leaf margins, rather than to olfactory signals. Our study provides direct experimental evidence for aposematic coloration in red leaves.

Protective effect of an oral natural phytonutrient in recurrent vulvovaginal candidiasis: a 12-month study.

The aim of the present study is to assess the clinical efficacy of a phytocompound with antimicotic properties (K-712, with the following 100 mg composition: 10 mg of oleoresin from Pseudowintera colorata at 30 percent concentration in Polygodial together with trace amounts of Olea europea) in recurrent vulvo-vaginal candidiasis (RVVC) as compared to an azole drug during a 12-month period: 6 months of treatment followed by 6 months of observation. This prospective randomized study involved 82 women (19-61 years) with complaints of abnormal vaginal discharge and with a history of at least four proven episodes of RVVC in the previous 12 months. Patients were divided into two groups of treatment of 41 patients each and were given: A) Itraconazole 200 mg orally daily for 4 days, then 200 mg once weekly for 6 months or B) 1 tablet twice a day of a K-712 for 4 weeks and then for the first 2 weeks of each month for a total of 6 months. Both groups were then followed-up for further 6 months. Each treatment schedule was well tolerated with only 4 patients in the azole group complaining of transient mild symptoms (nausea, abdominal discomfort, unpleasant taste). Itraconazole reached an earlier symptomatic relief during the first two weeks of observation as compared with K-712 (p<0.05) but both treatments enabled a comparable benefit during the entire treatment study period, afterwards with comparable symptom/sign score (itraconazole vs K-712: 9 vs 11). At 6-month observation, mycological cure was reached by 83 percent in the itraconazole group and in 78 percent of the K-712-treated patients. During the further 6-month observation period without treatment, the itraconazole group showed significantly more relapses (65.7 vs 34.2 in K-712, p<0.05) and at the end of the whole 12-month study period the mycological cure was significantly higher in the K-712-treated patients (65.8 vs 34.3 percent, p<0.05). There was a non- significant trend increase of less drug-susceptible species in the itraconazole group. From these preliminary data it would appear that a natural antifungal phytocompound proves to be as good as itraconazole in the maintenance treatment of RVVC. Moreover, this approach seems to maintain a higher mycological success rate afterwards by reducing the number of relapses and probably of the growth of azole-resistant species.

Subclinical candiduria in patients with gastrointestinal malignancies: a preliminary study on the protective effect of a natural phytocompound.

There is a great concern for the increasing incidence of candidiasis in cancer patients following immune-suppressive, cytostatic or antibiotic treatment. There are cancer patients with repeat asymptomatic recovery of candida in the urine in whom the choice of treatment, if any, is still a matter of debate. The aim of the study is to test the efficacy and tolerability of a natural anti-fungal phytocompound in patients with tumors of the gastrointestinal tract with prior or ongoing candiduria. Thirty-nine patients with operated gastrointestinal malignancies (18 still under current chemotherapy) with a history of repeated candiduria were enrolled. Eleven patients showed candiduria on enrolment and were treated with K-712, a natural antifungal phytocompound. Genomic analysis was carried out on blood samples of all patients on a monthly basis for 6 months. Within 3 weeks all 11 treated patients had negative cultures in the urine (10 patients after 2 weeks), 7 patients remained free of candiduria throughout the study period while 4 required a new treatment course. Three patients had positive genomic tests for systemic candidiasis and were treated with fluconazole. Eighteen (64 percent) out of the 28 patients who were free of candiduria on enrolment, developed a urinary candida infection during the 6-month follow-up and all cases were successfully treated with K-712. Seven (38 percent) of these cases presented a further recurrence at a later stage and all responded to a new course of K-172. No positive genomic tests were observed during the follow-up period. These data suggest that a consistent part of patients, mostly with gastrointestinal malignancies develop urinary candida infection when following chemotherapy treatment. A therapeutic approach with a natural antifungal phytocompound seems a safe and effective measure and a tentative prophylactic approach might also be envisaged.

Chemotaxonomy of Pseudowintera: sesquiterpene dialdehyde variants are species markers.

Two sesquiterpene dialdehydes, the 1beta-E-coumaroyl-5alpha-hydroxypolygodial plus the known 1beta-E-cinnamoyl-5alpha-hydroxypolygodial, were isolated from the recently described species Pseudowintera insperata. This discovery is a further example of the rare sesquiterpene dialdehyde coumarate/cinnamate combination being found exclusively in the family Winteraceae. Another sesquiterpene dialdehyde, isopaxidal, with the rare rearranged drimane skeleton, was isolated from Pseudowintera axillaris. The sesquiterpene dialdehyde contents of leaves of 25 individual plants of the four Pseudowintera species, all endemic to New Zealand, were measured by HPLC. P. insperata individuals all had high levels (3.0-6.9% of leaf dry wt.) of the coumarate, P. axillaris had high levels (2.2-6.9%) of paxidal, and Pseudowintera colorata from different areas of New Zealand contained varying levels of polygodial (1.4-2.9%) and 9-deoxymuzigadial (0-2.9%). Therefore the sesquiterpene dialdehydes are good species markers.

Fungicidal sesquiterpene dialdehyde cinnamates from Pseudowintera axillaris.

Bioactivity-directed separation of a foliage extract from the New Zealand shrub Pseudowintera axillaris led to a compound with fungicidal activity against the plant pathogen Phytophthora infestans. This was identified as a new sesquiterpene dialdehyde cinnamate named paxidal. Two 6-hydroxy derivatives were present at lower levels in the extract. A further nine derivatives were synthesized from these natural products for a structure-activity study against a range of important food crop pathogens. The cinnamate group was important for fungicidal effects, and protection of the dialdehyde as a dimethyl acetal gave more potent, broader spectrum activity.

Do carbon-based defences reduce foliar damage? Habitat-related effects on tree seedling performance in a temperate rainforest of Chiloé Island, Chile.

Carbon-based secondary compounds (CBSCs), such as phenols or tannins, have been considered as one of the most important and general chemical barriers of woody plants against a diverse array of herbivores. Herbivory has been described as a critical factor affecting the growth and survival of newly established tree seedlings or juveniles then, the presence of secondary metabolites as defences against herbivores should be a primary strategy to reduce foliar damage. We examined whether light-induced changes in leaf phenolic chemistry affected insect herbivory on seedlings of two rainforest tree species, Drimys winteri (Winteraceae) and Gevuina avellana (Proteaceae). Seedlings of both species were planted under closed canopy and in a canopy gap within a large remnant forest patch. Half of the seedlings in each habitat were disinfected with a wide-spectrum systemic insecticide and the other half were used as controls. Seedling growth, survival, and foliar damage (estimated by an herbivory index) due to insect herbivores were monitored over a period of 16 months (December 2001-April 2003). The total leaf content of phenols and condensed tannins were assessed in seedlings from both habitats. As expected, access to light induced a greater production of CBSCs in seedlings of both tree species, but these compounds did not seem to play a significant defensive role, as seedlings grown in gaps suffered greater leaf damage than those planted in forest interior. In addition, in both habitats, seedlings without insecticide treatment suffered a greater foliar damage than those with insecticide, especially 16 months after the beginning of the experiment. Canopy openness and herbivory had positive and negative effects, respectively, on seedling growth and survival in both tree species. In conclusion, despite the higher levels of defence in tree-fall gap, the higher densities of herbivore override this and lead to higher damage levels.

Evidence for ovarian self-incompatibility as a cause of self-sterility in the relictual woody angiosperm, Pseudowintera axillaris (Winteraceae).

Species within the genus Pseudowintera exhibit high rates of self-sterility. Self-sterility in the genus has been previously posited-but not confirmed-to be the result of late-acting ovarian self-incompatibility (OSI) functioning within nucellar tissue of the ovule to prevent self pollen tubes from entering the embryo sac. Structural and functional aspects of pollen-carpel interactions and early seed development following cross- and self-pollination were investigated in P. axillaris to determine the site, timing and possible mechanisms of self-sterility. No significant differences were observed between pollen tube growth, ovule penetration and double fertilization following cross- and self-pollination. Pollen tubes exhibited phasic growth in an extracellular matrix composed of proteins and carbohydrates, as well as arabinogalactans/arabinogalactan proteins. A uniform failure in embryo sac development prior to division of the zygote was apparent within 15 d following double fertilization by self gametes. Results indicate that SI mechanisms in P. axillaris do not prevent double fertilization from occurring. Instead, mechanisms of self-sterility affect post-zygotic development of the embryo sac. Although self-sterility may be attributed to inbreeding depression, given the post-zygotic nature of failure in embryo sac development, the possibility of late-acting OSI is discussed.