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.

Infection with Menangle virus in flying foxes (Pteropus spp.) in Australia

Objective: To examine flying foxes (Pteropus spp.) for evidence of infection with Menangle virus. Design: Clustered non-random sampling for serology, virus isolation and electron microscopy (EM). Procedure: Serum samples were collected from 306 Pteropus spp. in northern and eastern Australia and tested for antibodies against Menangle virus (MenV) using a virus neutralisation test (VNT). Virus isolation was attempted from tissues and faeces collected from 215 Pteropus spp. in New South Wales. Faecal samples from 68 individual Pteropus spp. and four pools of faeces were examined by transmission EM following routine negative staining and immunogold labelling. Results: Neutralising antibodies (VNT titres ≥ 8) against MenV were detected in 46% of black flying foxes (P. alecto), 41% of grey-headed flying foxes (P. poliocephalus), 25% of spectacled flying foxes (P. conspicillatus) and 1% of little red flying foxes (P. scapulatus) in Australia. Positive sera included samples collected from P. poliocephalus in a colony adjacent to a piggery that had experienced reproductive disease caused by MenV. Virus-like particles were observed by EM in faeces from Pteropus spp. and reactivity was detected in pooled faeces and urine by immunogold EM using sera from sows that had been exposed to MenV. Attempts to isolate the virus from the faeces and tissues from Pteropus spp. were unsuccessful. Conclusion: Serological evidence of infection with MenV was detected in Pteropus spp. in Australia. Although virus-like particles were detected in faeces, no viruses were isolated from faeces, urine or tissues of Pteropus spp.

Laboratory studies on Australian isolates of Metarhizium anisopliae as a biopesticide for the cattle tick Boophilus microplus

Thirty-one isolates of Metarhizium anisopliae were bioassayed against the cattle tick (Boophilus microplus). More than half of the isolates showed a high degree of virulence to ticks. Radial growth curves for growth between 20 °C and 40 °C were obtained for all isolates. This information together with information on virulence will be important for the selection of isolates suitable to kill ticks on the surface of cattle. A biopesticide for cattle ticks must kill ticks rapidly at temperatures within the upper end of most isolates' growth curves. It was also found that the time taken to achieve 100% tick mortality in vitro using a virulent isolate could be halved by applying conidia in a 10% oil emulsion. Scanning electron microscopy and light microscopy were used to investigate and compare the germination and penetration of conidia formulated in aqueous and oil formulations. It was found that conidia in both formulations were able to germinate and produce appressoria on the surface of ticks in less than 11 h. Marked weakness within 26 h, followed by extensive hyphal growth on the cuticle characterised the invasion of ticks by M. anisopliae.

Identification of two distinct bovine parainfluenza virus type 3 genotypes

The partial gene sequencing of the matrix (M) protein from seven clinical isolates of bovine parainfluenza virus type 3 (BPIV-3), and the complete sequencing of a representative isolate (Q5592) was completed in this study. Nucleotide sequence analysis was initiated because of the failure of in-house BPIV-3 RT-PCR methods to yield expected products for four of the isolates. Phylogenetic reconstructions based on the nucleotide sequences for the M-protein and the entire genome, using all of the available BPIV-3 nucleotide sequences, demonstrated that there were two distinct BPIV-3 genotypes (BPIV-3a and BPIV-3b). These newly identified genotypes have implications for the development of BPIV-3 molecular detection methods and may also impact on BPIV-3 vaccine formulations.

DNA amplification fingerprinting analysis of genetic variation within Fusarium oxysporum f.sp zingiberi

Genetic variation among 29 isolates of Fusarium oxysporum f.sp. zingiberi (Foz) collected from diseased ginger rhizome in production regions throughout Queensland was analysed using DNA amplification fingerprinting (DAF). Eight isolates of other Fusarium species and/or formae speciales were included for comparative analysis. Within the Foz isolates, three haplotypes were identified based on 17 polymorphic bands generated with five primers. Two groups showed very little genetic variation (98.6% similarity), whereas the third single isolate was quite distinct in terms of its molecular profile (77.2% similarity). Genetic similarity among the Fusarium solani, F. oxysporum f.sp. lycopersici and F. oxysporum f.sp. cubense races 1, 3 and 4 isolates compared well with the published literature.

Capsicum chlorosis virus infecting Capsicum annuum in the East Kimberley region of Western Australia

Capsicum chlorosis virus (CaCV) was detected in field grown Capsicum annuum from Kununurra in northeast Western Australia. Identification of the Kununurra isolate (WA-99) was confirmed using sap transmission to indicator hosts, positive reactions with tospovirus serogroup IV-specific antibodies and CaCV-specific primers, and amino acid sequence comparisons that showed >97% identity with published CaCV nucleocapsid gene sequences. The reactions of indicator hosts to infection with WA-99 often differed from those of the type isolate from Queensland. The virus multiplied best when test plants were grown at warm temperatures. CaCV was not detected in samples collected in a survey of C. annuum crops planted in the Perth Metropolitan area.

A detached leaf bioassay to screen Durian cultivars for susceptibility to Phytophthora palmivora

A detached-leaf bioassay was developed and used to screen five durian (Durio zibethinus) cultivars against Phytophthora palmivora isolates from a trunk canker, root and fruit. The fruit isolate was less aggressive than the canker and root isolates. The bioassay using the canker isolate was later used to determine the variation in resistance of D. macarantha and nineteen cultivars of D. zibethinus. The cultivars displayed a range of responses with Parung and Gob being most tolerant, with Gaan Yaow, Chanee and Penang 88 being susceptible. The remaining germplasm fell between Gaan Yaow and Penang 88 in susceptibility. The leaf bioassay was found to be a rapid and reliable method for assessing the susceptibility of durian cultivars.

Phylogenetic relationships of unclassified, satellitic Pasteurellaceae obtained from different species of birds as demonstrated by 16S rRNA gene sequence comparison

Avian haemophili demonstrating in vitro satellitic growth, also referred to as the V-factor or NAD requirement, have mainly been classified with Avibacterium paragallinarum (Haemophilus paragallinarum), Avibacterium avium (Pasteurella avium), Avibacterium volantium (Pasteurella volantium) and Avibacterium sp. A (Pasteurella species A). The aim of the present study was to assess the taxonomic position of 18 V-factor-requiring isolates of unclassified Haemophilus-like organisms isolated from galliforme, anseriforme, columbiforme and gruiforme birds as well as kestrels and psittacine birds including budgerigars by conventional phenotypic tests and 16S rRNA gene sequencing. All isolates shared phenotypical characteristics which allowed classification with Pasteurellaceae. Haemolysis of bovine red blood cells was negative. Haemin (X-factor) was not required for growth. Maximum-likelihood phylogenetic analysis including bootstrap analysis showed that six isolates were related to the avian 16S rRNA group and were classified as Avibacterium according to 16S rRNA sequence analysis. Surprisingly, the other 12 isolates were unrelated to Avibacterium. Two isolates were unrelated to any of the known 16S rRNA groups of Pasteurellaceae. Two isolates were related to Volucribacter of the avian 16S rRNA group. Seven isolates belonged to the Testudinis 16S rRNA group and out of these, two isolates were closely related to taxa 14 and 32 of Bisgaard, whereas four other isolates were found to form a genus-like group distantly related to taxon 40 and one isolated remained distantly related to other members of the Testudinis group. One isolate was closely related to taxon 26 (a member of Actinobacillus sensu stricto). The study documented major genetic diversity among V-factor-requiring avian isolates beyond the traditional interpretation that they only belong to Avibacterium, underlining the limited value of satellitic growth for identification of avian members of Pasteurellaceae. Our study also emphasized that these organisms will never be isolated without the use of special media satisfying the V-factor requirement.

Phylogenetic analysis of betanodavirus isolates from Australian finfish

In Australia, disease caused by betanodavirus has been reported in an increasing number of cultured finfish since the first report of mortalities in 1990. Partial coat protein gene sequences from the T2 or T4 regions of 8 betanodaviruses from barramundi Lates calcarifer, sleepy cod Oxyeleotris lineolata, striped trumpeter Latris lineata, barramundi cod Cromileptes altivelis, Australian bass Macquaria novemaculata and gold-spotted rockcod Epinephelus coioides from several Australian states were determined. Analysis of the 606 bp nucleotide sequences of the T2 region of 4 isolates demonstrated the close relationship with isolates from the red-spotted grouper nervous necrosis virus (RGNNV) genotype and the Cluster Ia subtype. Comparison of a smaller 289 bp sequence from the T4 region identified 2 distinct groupings of the Australian isolates within the RGNNV genotype. Isolates from barramundi from the Northern Territory, barramundi, sleepy cod, barramundi cod and gold-spotted rockcod from Queensland, and striped trumpeter from Tasmania shared a 96.2 to 99.7%, nucleotide identity with each other. These isolates were most similar to the RGNNV genotype Cluster Ia. Isolates from Australian bass from New South Wales and from barramundi from South Australia shared a 98.6% sequence identity with each other. However, these isolates only shared an 85.8 to 87.9%, identity with the other Australian isolates and representative RGNNV isolates. The closest nucleotide identity to sequences reported in the literature for the New South Wales and South Australian isolates was to an Australian barramundi isolate (Ba94Aus) from 1994. These 2 Australian isolates formed a new subtype within the RGNNV genotype, which is designated as Cluster Ic.

First report of Colombian datura virus from Australia.

Colombian datura virus was identified from the ornamental plant Brugmansia sp., showing leaf mosaic symptoms. The nucleotide sequence of the 3 untranslated region and the amino acid sequence of the 3 portion of the coat protein were 100% identical to those from a Hungarian isolate of the virus. This represents the first record of this virus in Australia.

Austropleospora osteospermi gen. et sp nov and its host specificity and distribution on Chrysanthemoides monilifera ssp rotundata in Australia.

Hendersonia osteospermi was found for the first time in Australia on leaf spots of the introduced invasive plant Chrysanthemoides monilifera ssp. rotundata (bitou bush) in coastal regions of New South Wales. Pathogenicity tests on species from 11 tribes in the family Asteraceae, demonstrated that H. osteospermi caused severe necrosis on leaves and stems of C. monilifera ssp. rotundata and its congener C. monilifera ssp. monilifera (boneseed). Small necrotic spots also developed on Osteospermum fruticosum and Dimorphotheca cuneata in the Calenduleae and on Helianthus annuus (sunflower) in the Heliantheae. None of the other plant species tested developed leaf spots, although H. osteospermi was re-isolated from senescent leaves of Cynara scolymus (globe artichoke) in the Cynareae and Vernonia cinerea in the Vernonieae. Single ascospores from ascomata of a Pleospora-like fungus found on diseased stems of bitou bush produced H. osteospermi in culture, which proved the anamorph/teleomorph connection. The ITS region of both a single-ascospore isolate and a single-conidium isolate were sequenced and found to be identical. The taxonomic status of H. osteospermi is re-examined and Austropleospora osteospermi gen. et sp. nov. is described as its teleomorph based on morphology, host range tests and DNA sequence analysis. The potential of A. osteospermi for the biological control of bitou bush and boneseed in Australia is discussed.

Characterization of highly informative cross-species microsatellite panels for the Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) including five novel primers

The Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) are threatened species of aquatic mammals in the order Sirenia. Sirenian conservation and management actions would benefit from a more complete understanding of genetic diversity and population structure. Generally, species-specific microsatellite markers are employed in conservation genetic studies; however, robust markers can be difficult and costly to isolate. To increase the number of available markers, dugong and manatee microsatellite primers were evaluated for cross-species amplification. Furthermore, one manatee and four dugong novel primers are reported. After polymerase chain reaction optimization, 23 (92%) manatee primers successfully amplified dugong DNA, of which 11 (48%) were polymorphic. Of the 32 dugong primers tested, 27 (84%) yielded product in the manatee, of which 17 (63%) were polymorphic. Dugong and manatee primers were compared and the most informative markers were selected to create robust and informative marker-panels for each species. These cross-species microsatellite marker-panels can be employed to assess other sirenian populations and can provide beneficial information for the protection and management of these unique mammals.

Using bacteria in banana for efficient use of fertilizer

This project aims to develop sustainable banana production practices by improving efficiency of fertilizer use. We will investigate how we can reintroduce endophytic beneficial bacteria that can be established inside the banana host to provide lasting and durable benefits to growth. In the current research we have been able to isolate bacteria from inside banana and preliminary characterisation indicates isolates with a range of attributes for improved efficiency of fertilizer uptake. Experimentation will include evaluation in vitro and pot trials and field trials using bacteria most likely to increase plant access to nutrients as well to compare nutrient impacts from conventional verses slow release fertilizer.

Unusual growth variants of Avibacterium paragallinarum.

Two isolates of haemophilic bacteria originally isolated in the 1980s from chickens were re-examined. The addition of a 10% sterile filtrate from an overnight culture of Staphylococcus epidermidis allowed growth of both isolates in solid and liquid media that were otherwise not capable of supporting the growth of these isolates. Using the modified media, genotypic and serotypic studies were performed, which confirmed both isolates to be Avibacterium paragallinarum, with one isolate being serovar A and the other serovar C. The unusual growth requirements of these two isolates reinforces the need for careful interpretation by diagnostic laboratories examining chickens showing signs of upper respiratory tract disease.

Comparative studies on the invasion of cattle ticks (Rhipicephalus (Boophilus) microplus) and sheep blowflies (Lucilia cuprina) by Metarhizium anisopliae (Sorokin).

Microscopic investigations over time were carried out to study and compare the pathogenesis of invasion of ticks and blowflies by Metarhizium anisopliae. The scanning electron microscope and stereo light microscope were used to observe and record processes on the arthropods' surfaces and the compound light microscope was used to observe and record processes within the body cavities. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle, particularly the outer endocuticle. Although large numbers of conidia adhered to the surface of blowflies, no conidia were seen to germinate on external surfaces. A single germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggests that fungal invasion of the blowfly is primarily through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on tick cuticles, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16.