Nitrogen fixation associated with sago (Metroxylon sagu) and some implications.

AIMS: To determine the presence and contribution of diazotrophic bacteria to nitrogen concentrations in edible starch derived from the sago palm (Metroxylon sagu). METHODS AND RESULTS: Isolation of diazotrophic bacteria and analysis of nitrogen fixation were conducted on pith, root and sago starch samples. Acetylene reduction showed that five of ten starch samples were fixing nitrogen. Two presumptive nitrogen-fixing bacteria from starch fixed nitrogen in pure culture and five isolates were positive for the nif H gene. Nitrogen concentrations in 51 starch samples were low (37 samples <0·2 g kg(-1); 14 ranging from 0·2 to 2·0 g kg(-1)). CONCLUSIONS: Nitrogen fixation occurs in sago starch, which undoubtedly plays a role in fermentation ecology. Nitrogen levels are considered too low to be of nutritional benefit and to protect against nutritional-associated illnesses. SIGNIFICANCE AND IMPACT OF THE STUDY: Sago starch does not add significantly to the protein calorie intake and may be associated with susceptibility to nutritional-associated illness.

Spontaneous fermentation of traditional sago starch in Papua New Guinea.

Sago starch is an important dietary carbohydrate in lowland Papua New Guinea (PNG). An investigation was conducted to determine whether microbes play a role in its preservation using traditional methods. In 12 stored sago samples collected from PNG villages, lactic acid bacteria (LAB) were present (> or = 3.6 x 10(4)cfu/g) and pH ranged from 6.8 to 4.2. Acetic and propionic acids were detected in all samples, while butyric, lactic and valeric acids were present in six or more. In freshly prepared sago, held in sealed containers in the laboratory at 30 degrees C, spontaneous fermentation by endogenous microflora of sago starch was observed. This was evident by increasing concentrations of acetic, butyric and lactic acids over 4 weeks, and pH reducing from 4.9 to 3.1: both LAB and yeasts were involved. Survival of potential bacterial pathogens was monitored by seeding sago starch with approximately 10(4)/g of selected organisms. Numbers of Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus fell to < 30/g within 7 days. Salmonella sp. was present only in low numbers after 7 days (< 36/g), but Escherichia coli was still detectable after three weeks (> 10(2)/g). Fermentation appeared to increase the storability and safety of the product.

Mycotoxins and toxigenic fungi in sago starch from Papua New Guinea.

AIMS: To assay sago starch from Papua New Guinea (PNG) for important mycotoxins and to test fungal isolates from sago for mycotoxin production in culture. METHODS AND RESULTS: Sago starch collected from Western and East Sepik Provinces was assayed for aflatoxins, ochratoxin A, cyclopiazonic acid, sterigmatocystin, citrinin and zearalenone and all 51 samples were negative. Frequently isolated species of Penicillium (13), Aspergillus (five) and Fusarium (one) were cultured on wheat grain, and tested for the production of ochratoxin A, cyclopiazonic acid, sterigmatocystin, citrinin, patulin and penicillic acid. All 12 isolates of P. citrinin and one of two A. flavipes isolates produced citrinin. A single isolate of A. versicolor produced sterigmatocystin. No other mycotoxins were detected in these cultures. CONCLUSIONS: No evidence was found of systemic mycotoxin contamination of sago starch. However, the isolation of several mycotoxigenic fungi shows the potential for citrinin and other mycotoxins to be produced in sago stored under special conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Sago starch is the staple carbohydrate in lowland PNG and the absence of mycotoxins in freshly prepared sago starch is a positive finding. However, the frequent isolation of citrinin-producing fungi indicates a potential health risk for sago consumers, and food safety is dependant on promoting good storage practices.

Bacterial contamination of sago starch in Papua New Guinea.

Sago starch is an important food in lowland Papua New Guinea. Extraction of the starch from the palm and storage were performed by way of traditional methods that have been used for thousands of years. Currently, very little is known about the microbiology of sago starch. Sago samples were collected from areas of high starch utilization and analyzed for the presence of bacterial pathogens and indicator organisms. Storage methods and duration were recorded at the time of collection, and pH and water activity on arrival at the laboratory. Sago starch was found to harbor high levels of fecal contamination, as well as various food pathogens including Salmonella, Bacillus cereus, and coagulase-positive staphylococci. Clostridium perfringens was only present infrequently in samples and in very low numbers, while Listeria monocytogenes was not isolated from sago starch. The presence of high levels of fecal contamination in sago starch is of particular concern, and may contribute to diarrheal disease in rural Papua New Guinea.

Fungal colonization of sago starch in Papua New Guinea.

Sago starch is an important source of dietary carbohydrates in lowland Papua New Guinea. Over the past 30 years there have been sporadic reports of severe illness following consumption of sago starch. A common assumption is that fungal metabolites might be associated with the illness, leading to the need for a more thorough investigation of the mycoflora of sago starch. Sago starch was collected from areas of high sago consumption in Papua New Guinea for fungal analysis (69 samples). Storage methods and duration were recorded at the time of collection and pH on arrival at the laboratory. Yeasts were isolated from all samples except two, ranging from 1.2 x 10(3) to 8.3 x 10(7) cfu/g. Moulds were isolated from 65 of the 69 samples, ranging from 1.0 x 10(2) to 3.0 x 10(6) cfu/g. Of 44 samples tested for ergosterol content, 42 samples showed the presence of fungal biomass. Statistical analyses indicated that sago starch stored for greater than five weeks yielded significantly higher ergosterol content and higher numbers of moulds than sago stored for less than five weeks. The method of storage was also shown to influence mould numbers with storage in natural woven fibre containers returning significantly greater numbers than present in other storage methods tested. Potentially mycotoxigenic genera of moulds including Aspergillus and Penicillium were commonly isolated from sago starch, and as such storage factors that influence the growth of these and other filamentous fungi might contribute to the safety of traditional sago starch in PNG.

Bacterial flora from the gut of the wild and cultured banana prawn, Penaeus merguiensis.

AIMS: There is growing awareness of the influence of the bacterial composition of the gut on the health and growth of the host. This study compared the bacterial flora from the digestive system of the wild and cultured prawn, Penaeus merguiensis. METHODS AND RESULTS: Whole guts were dissected from wild and cultured prawns and divided into sections corresponding to the foregut, digestive gland, midgut and hindgut. Homogenates of these sections were plated onto seawater nutrient agar and the colonies identified to genus level and, in some cases, species. Quantitative and qualitative comparisons amongst gut regions for both wild and cultured prawns are presented. CONCLUSIONS: Both wild and cultured prawns supported remarkably similar bacterial floral compositions, which included members from the genera Aeromonas, Plesiomonas, Photobacterium, Pseudoalteromonas, Pseudomonas and Vibrio. Members of the genus Vibrio were quantitatively dominant. A number of Vibrio species were recovered solely from cultured prawns. Of these, Vibrio gazogenes was the most notable (numerically dominating in all but the midgut). The opportunistic pathogen V. parahaemolyticus was also recovered. SIGNIFICANCE AND IMPACT OF THE STUDY: The remarkable similarity of gut compositions between wild and cultured prawns, despite being drawn from very different habitats, suggests an influence of the host on the establishment of the gut flora. An understanding of host/gut floral interactions has significance in fostering conditions which promote the growth of cultivated hosts.

Survival of Aedes aegypti (Diptera: Culicidae) eggs in surface and subterranean breeding sites during the northern Queensland dry season.

The effect of a protracted dry season on the viability of Ae. aegypti (L.) eggs was examined in Townsville, northern Queensland, Australia. Eggs were placed in several different surface and subterranean larval habitats; and after four dry season months, only 1-10% of eggs remained viable in the surface and subterranean sites, respectively. Low humidity and predation by Periplaneta americana (L.) were the major causes of egg mortality in eggs in surface sites. P. americana was the most significant cause of egg predation in subterranean breeding sites but fungi, especially Penicillium citrinum Thom, covered egg batches within 15 d. Mycotoxins produced by the spores of P. citrinum are believed to have killed embryonating eggs. The high mortality rate of Ae. aegypti eggs during the dry season suggests that this survival strategy is unlikely to contribute to rapid and successful recolonization of surface sites at the end of the wet season.

Mucor amphibiorum in the toad, Bufo marinus, in Australia.

Mucor amphibiorum, a fungus previously found in captive amphibians in Europe and the platypus in Australia, was observed in free-ranging toads, Bufo marinus, in Australia. In tissues the fungus occurred as sphaerules 4.9 to 36.4 microns in diameter; hyphae were not formed. Some spharules developed two to 11 daughter sphaerules internally and these were released into tissues by dissolution of the outer wall. Infected toads were found at 11 sites from nine locations in northern and eastern Australia. The overall prevalence of infection in 3,518 toads was 0.71%. Mucor amphibiorum was isolated from soil at one location.

The origin and pathogenicity of Basidiobolus species in northern Australia.

Basidiobolus haptosporus Drechsler causes human and animal disease in the tropics. This paper reports the isolation of this organism from natural substrates and describes the pathogenicity of the isolates. Basidiobolus was recovered from faecal samples of amphibians, reptiles and macropods, from woodlice, and from granulomatous skin lesions of horses. Some isolates were heat-tolerant. Almost all of the heat-tolerant isolates were pathogenic to suckling mice and had smooth or undulate, or smooth plus undulate zygospore walls. When inoculated intracerebrally into suckling mice they caused encephalomalacia, necrosis, meningoencephalitis, congestion, haemorrhage and a granulomatous reaction in the central nervous tissue, hydrocephalus and nervous signs. There was a variety of animal sources of pathogenic strains of the organism which were widely distributed in the sampling area, thus the potential hazard of infection could be more serious than previously considered.

Sporulation media for Basidiobolus species.

Basidiobolus species lose their sporulation ability after relatively short periods of time in culture. This problem has been partially overcome by the use of media which incorporate glucosamine hydrochloride and casein hydrolysate. The media also appear to be suitable for the culture of Conidiobolus species.

Pythium destruens sp. nov., an agent of equine pythiosis.

The equine phycomycotic agent known commonly as Hyphomyces destruens or occasionally as Pythium gracile, is described as a new species Pythium destruens. Separation is on the basis of morphological features, temperature growth profiles, esterase/lipase activity, and carbohydrate utilization ability. P. diclinum (synonymous with P. gracile sensu Middleton) showed minor differences in vesicle, oospore and oogonium size from P. destruens. P. destruens grew at 40 degrees C on corn meal agar and hydrolysed esters of lauric and oleic acids. These abilities were not displayed by P. diclinum, but this species grew more vigorously on cellobiose, fructose, gentibiose, inulin, raffinose, maltose, mannose, salicin, starch and sucrose than P. destruens. The latter species showed no substantial ability for growth on inulin, raffinose, and salicin. Equine isolates from Australia, Japan and New Guinea were similar.

Clonal propagation of Leptospermum spp. by tissue culture.

Propagation by axillary and multiple axillary bud development was achieved in three native Leptospermum spp. when axillary buds derived from nodal tissues ex mature plants were placed in benzylaminopurine media (0.04-1.0 µM) containing macro- and micro-nutrients, sucrose (0.06 M) and a vitamin/amino acid supplement. Reduction of agar concentration from 0.8 to 0.2% greatly stimulated axillary bud development and growth in L. flavescens and L. brachyandrum. Rooting of axillary shoots was stimulated by 2,4-dichlorophenoxyacetic acid and p-chlorophenoxy acetic acid in L. flavescens at concentrations of 5 and 1 µM respectively. In L. petersonii ssp. root initiation and development was favoured by ß-naphthoxyacetic acid (1 µM) and in L. brachyandrum indole butyric acid and α-naphthalene acetic acid (1 µM) were almost equally effective.

Clonal propagation of Callistemon by tissue culture techniques.

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 µM), nicotinic acid (20 µM), pyridoxine hydrochloride (3 µM), thiamine hydrochloride (2 µM), riboflavin (10 µM), cytokinins (5 µM) and auxins (0.1 µM). The presence of benzylaminopurine (5 µM) and p-chlorophenoxyacetic acid (0.1 µM) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 µM. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 µM.

Halteromyces, a new genus in the Mucorales.

Halteromyces radiatus is described as a new genus and species in the order Mucorales. The genus is referred to the family Mucoracease and has close affinities to the genus Absidia. The fungus grows readily in pure culture. The asexual structures are described; no sexual structures have been found despite extensive contrasts with members of the genus Absidia and Gongronella.