In vitro biological control of Pyrrhoderma noxium using volatile compounds produced by termite gut-associated streptomycetes.

Introduction: Pyrrhoderma noxium is a plant pathogen that causes economic losses in agricultural and forestry industries, including significant destruction to amenity trees within the city of Brisbane in Australia. Use of chemical control agents are restricted in public areas, there is therefore an urgent need to investigate biological control approaches. Members of the phylum Actinomycetota, commonly known as actinomycetes, are known for their industrially important secondary metabolites including antifungal agents. They have proven to be ideal candidates to produce environmentally friendly compounds including the volatile organic compounds (VOCs) which can be used as biofumigants. Methods: Different Streptomyces species (n=15) previously isolated from the guts of termites and stored in the University of the Sunshine Coast'sMicrobial Library were tested for their antifungal VOCs against Pyrrhoderma noxium. Results: Fourteen of them were found to display inhibition (39.39-100%) to the mycelial development of the pathogen. Strongest antifungal activity displaying isolates USC-592, USC-595, USC-6910 and USC-6928 against the pathogen were selected for further investigations. Their VOCs were also found to have plant growth promotional activity observed for Arabidopsis thaliana with an increase of root length (22-36%) and shoot length (26-57%). The chlorophyll content of the test plant had a slight increase of 11.8% as well. Identified VOCs included geosmin, 2-methylisoborneol, 2-methylbutyrate, methylene cyclopentane, ß-pinene, dimethyl disulfide, ethyl isovalerate, methoxyphenyl-oxime and α-pinene. Additionally, all 15 Streptomyces isolates were found to produce siderophores and indole acetic acid as well as the enzyme chitinase which is known to break down the fungal cell wall. Discussion: Findings indicate that termite gut-associated streptomycetes might be used to control Pyrrhoderma noxium by utilizing their wide range of inhibitory mechanisms.

Identification of Acetomycin as an Antifungal Agent Produced by Termite Gut-Associated Streptomycetes against Pyrrhoderma noxium.

Plant fungal pathogen Pyrrhoderma noxium is responsible for the destructive and invasive disease of brown root rot currently affecting the city of Brisbane, Australia. In order to address this issue, environmentally friendly and safe alternatives to chemical control are preferred due to the city's public setting. Antifungal natural products are ideal candidates as biological control alternatives and can be detected through investigating the metabolomes of microbial symbionts. Within this study, an NMR-based metabolomics approach was applied to fermentation extracts obtained from 15 termite gut-associated streptomycetes. By analysing the NMR spectra, six of the extracts which displayed similar chemical profiles exhibited antifungal activity against the P. noxium pathogen. The major compound within these extracts was identified as acetomycin using NMR and X-ray crystallography analyses. This is the first reporting of acetomycin as a potential natural product fungicide, particularly as an antifungal agent against P. noxium. Inhibitory activity was also found against other important fungal crop pathogens, including Aspergillus niger, Botrytis cinerea, and Alteranaria alternata. Further experimentation using a woodblock test found inhibitory activity on the growth of the P. noxium pathogen for up to 3 weeks and a significant difference in the integrity of the woodblocks when conducting compression strength tests after 6 weeks. Therefore, acetomycin may be used as a biological control agent and natural product fungicide against P. noxium.

Untargeted MS-Based Metabolomic Analysis of Termite Gut-Associated Streptomycetes with Antifungal Activity against Pyrrhoderma noxium.

Pyrrhoderma noxium is a plant fungal pathogen that induces the disease of brown root rot in a large variety of tree species. It is currently infecting many of the amenity trees within Brisbane City of Queensland, Australia. Steering away from harmful chemical fungicides, biological control agents offer environmentally friendly alternatives. Streptomycetes are known for their production of novel bioactive secondary metabolites with biocontrol potential, particularly, streptomycete symbionts isolated from unique ecological niches. In this study, 37 termite gut-associated actinomycete isolates were identified using molecular methods and screened against P. noxium. A majority of the isolates belonged to the genus Streptomyces, and 15 isolates exhibited strong antifungal activity with up to 98.5% mycelial inhibition of the fungal pathogen. MS/MS molecular networking analysis of the isolates' fermentation extracts revealed several chemical classes with polyketides being among the most abundant. Most of the metabolites, however, did not have matches to the GNPS database, indicating potential novel antifungal compounds in the active extracts obtained from the isolates. Pathway enrichment and overrepresentation analyses revealed pathways relating to polyketide antibiotic production, among other antibiotic pathways, further confirming the biosynthetic potential of the termite gut-associated streptomycetes with biocontrol potential against P. noxium.

Nocardia australiensis sp. nov. and Nocardia spumae sp. nov., isolated from sea foam in Queensland, Australia.

Strains USC-21046T and USC-21048T were isolated from foaming coastal marine waters on the Sunshine Coast, Queensland, Australia. Both strains displayed growth and morphological characteristics typical for members belonging to the genus Nocardia. The major polar lipids were diphosphatidylglycerol and phosphatidylethanolamine, and the major fatty acids were C16 : 0, C18 : 1 ω9c, C18 : 0 and C18 : 0 10-methyl. The mycolic acids of strains USC-21046T and USC-21048T consisted of chain lengths between 50-64 and 56-68, respectively. Moreover, both of those strains contained meso-diaminopimelic acid and ribose, arabinose, glucose and galactose as whole cell sugars. Based on the phylogenomic results, both strains belonged to the genus Nocardia with strain USC-21046T showing an 80.4 % genome similarity to N. vinacea NBRC 16497T and N. pseudovaccinii NBRC 100343T, whereas USC-21048T strain showed an 83.6 % genome similarity to N. aobensis NBRC 100429T. Both strains were delineated from their closely related relatives based on physiological (e.g. growth on sole carbon source) and chemotaxonomic (e.g. cellular fatty composition) differences. The digital DNA-DNA hybridization (dDDH) values between USC-21046T and USC-21048T and their closely related relatives were below the dDDH threshold value of ≤70 % used for the taxonomic classification of novel species status. The genome length of strains USC-21046T and USC-21048T were 6 878 863 and 7 066 978 bp, with G+C contents of 65.2 and 67.8 mol%, respectively. For the novel isolates, we propose the names Nocardia australiensis sp. nov. with the type strain USC-21046T (=DSM 111727T=NCCB 100867T) and Nocardia spumae sp. nov. with the type strain USC-21048T (=DSM 111726T=NCCB 100868T).

Assessing the Various Antagonistic Mechanisms of Trichoderma Strains against the Brown Root Rot Pathogen Pyrrhoderma noxium Infecting Heritage Fig Trees.

A wide range of phytopathogenic fungi exist causing various plant diseases, which can lead to devastating economic, environmental, and social impacts on a global scale. One such fungus is Pyrrhoderma noxium, causing brown root rot disease in over 200 plant species of a variety of life forms mostly in the tropical and subtropical regions of the globe. The aim of this study was to discover the antagonistic abilities of two Trichoderma strains (#5001 and #5029) found to be closely related to Trichoderma reesei against P. noxium. The mycoparasitic mechanism of these Trichoderma strains against P. noxium involved coiling around the hyphae of the pathogen and producing appressorium like structures. Furthermore, a gene expression study identified an induced expression of the biological control activity associated genes in Trichoderma strains during the interaction with the pathogen. In addition, volatile and diffusible antifungal compounds produced by the Trichoderma strains were also effective in inhibiting the growth of the pathogen. The ability to produce Indole-3-acetic acid (IAA), siderophores and the volatile compounds related to plant growth promotion were also identified as added benefits to the performance of these Trichoderma strains as biological control agents. Overall, these results show promise for the possibility of using the Trichoderma strains as potential biological control agents to protect P. noxium infected trees as well as preventing new infections.

Comparative genomics of Nocardia seriolae reveals recent importation and subsequent widespread dissemination in mariculture farms in the South Central Coast region, Vietnam.

Between 2010 and 2015, nocardiosis outbreaks caused by Nocardia seriolae affected many permit farms throughout Vietnam, causing mass fish mortalities. To understand the biology, origin and epidemiology of these outbreaks, 20 N. seriolae strains collected from farms in four provinces in the South Central Coast region of Vietnam, along with two Taiwanese strains, were analysed using genetics and genomics. PFGE identified a single cluster amongst all Vietnamese strains that was distinct from the Taiwanese strains. Like the PFGE findings, phylogenomic and SNP genotyping analyses revealed that all Vietnamese N. seriolae strains belonged to a single, unique clade. Strains fell into two subclades that differed by 103 SNPs, with almost no diversity within clades (0-5 SNPs). There was no association between geographical origin and subclade placement, suggesting frequent N. seriolae transmission between Vietnamese mariculture facilities during the outbreaks. The Vietnamese strains shared a common ancestor with strains from Japan and China, with the closest strain, UTF1 from Japan, differing by just 220 SNPs from the Vietnamese ancestral node. Draft Vietnamese genomes range from 7.55 to 7.96 Mbp in size, have an average G+C content of 68.2 % and encode 7 602-7958 predicted genes. Several putative virulence factors were identified, including genes associated with host cell adhesion, invasion, intracellular survival, antibiotic and toxic compound resistance, and haemolysin biosynthesis. Our findings provide important new insights into the epidemiology and pathogenicity of N. seriolae and will aid future vaccine development and disease management strategies, with the ultimate goal of nocardiosis-free aquaculture.

Testing the Biocontrol Ability of a Trichoderma-Streptomycetes Consortium against Pyrrhoderma noxium (Corner) L.W. Zhou and Y.C. Dai in Soil.

The Brown root rot pathogen Pyrrhoderma noxium (Corner) L.W. Zhou and Y.C. Dai is known to infect a large number of culturally and economically important plant species across the world. Although chemical control measures have been effective in managing this pathogen, their adverse effects on the ecosystem have limited their use. The use of biological control agents (BCAs) thus is generally accepted as an environmentally friendly way of managing various pathogens. Testing various consortia of the BCAs with different antagonistic mechanisms may even provide better disease protection than the use of a single BCA against aggressive plant pathogens such as the P. noxium. In the presented study, the wood decay experiment and the pot trial confirmed that the consortium of Trichoderma strains (#5029 and 5001) and streptomycetes (#USC-6914 and #USC-595-B) used was effective in protecting wood decay and plant disease caused by P. noxium. Among the treatments, complete elimination of the pathogen was observed when the BCAs were applied as a consortium. In addition, the BCAs used in this study promoted the plant growth. Therefore, Trichoderma and streptomycetes consortium could be used as a potential biocontrol measure to manage P. noxium infections in the field over the application of hazardous chemical control measures.

Isolation and Characterization of Nocardiae Associated with Foaming Coastal Marine Waters.

Nocardiosis is an infectious disease caused by Nocardia species that occurs worldwide, albeit more prevalently in tropical/subtropical regions. It can appear as either acute, subacute or as a chronic infection mostly with those with a compromised/weakened immune system. Inhalation of spores and or mycelium fragments is the main transmission route for developing pulmonary nocardiosis. In contrast, cutaneous nocardiosis usually occurs via direct contact. In the subtropical region of the Sunshine Coast in Australia foaming events with thick and persistent and orange-brown color foam have been observed during summer seasons in the near shore marine environments. This study reports the existence of nocardiae in these near shore marine environments by the use of a novel isolation method which used the gas requirements of nocardiae as a selective battery. A total of 32 nocardiae were isolated with the use of this novel method and subsequently conducted molecular identification methods confirmed that the isolates belonged to the genus Nocardia. Twenty-one isolates out of the 32 were closely related to N. nova strains MGA115 and one was related to CBU 09/875, in addition when compared with human pathogenic nocardiae twenty of the isolates were found to be related to N. nova strain JCM 6044. Isolates displayed varied resistance against some of the antibiotics tested when interpretation threshold recommended the Comite de L'Antibiogramme de la Societe Francaise de Microbiologie were used. The highest level of resistance against cefotaxime (n = 27) and ceftriaxone (n = 24). Some of the isolates (n = 6) that displayed resistance to selected antibiotics also possessed potential human pathogenic characteristics such as adherence and translocation through human long epithelial cells as well as displaying phage resistance (n = 26). They might thus present a potential public health risk if frequently encountered through exposure to aerosols generated by the foam as well as direct contact through a wound. Preventative measures to control the growth of nocardiae in such environments such as the control of pollutants, might prevent potential infections that might be caused by these bacteria in humans as well as in marine animals.

Bioactive Streptomycetes from Isolation to Applications: A Tasmanian Potato Farm Example.

The genus Streptomyces constitutes approximately 50% of all soil actinomycetes, playing a significant role in the soil microbial community through vital functions including nutrient cycling, production of bioactive metabolites, disease-suppression and plant growth promotion. Streptomyces produce many bioactive compounds and are prime targets for industrial and biotechnological applications. In addition to their agrobiological roles, some Streptomyces spp. can, however, be phytopathogenic, examples include, common scab of potato that causes economic losses worldwide. Currently used chemical control measures can have detrimental effect to environmental and human health as a result alternative methods to chemical disease control are being investigated. One alternative is the use of streptomycete specific phages to remove this pathogenic bacterium before it can cause the disease on potatoes. However, due to co-existence of non-common scab-causing species belonging to the genus Streptomyces, phage treatment is likely to affect a wide range of non-target streptomycete species including the beneficial ones in the soil. Therefore, before such treatment starts the host range of the phages within the targeted family of bacteria should be determined. In a study conducted using soil samples from a Tasmanian potato farm, streptomycetes were isolated and tested against streptomycete-specific phages. Their antifungal activity was also determined using multiple assays against selected phytopathogens. The four strongest antifungal activity-displaying isolates were further tested for their persistent antifungal activity using wheat and Fusarium solani in a pot trial. A second pot trial was also conducted to evaluate whether the beneficial streptomycetes were affected by streptophage treatment and whether their removal via the phage battery would cause opportunistic fungal infections to plants in soil. The streptomycetes prevented the reduction in wheat shoot weight caused by F. solani indicating their disease suppressive effect. However, when phages were added into the pots, the growth of wheat was detrimentally impacted. This finding might suggest that the reduced presence of antifungal streptomycetes via phage-induced lysis might encourage opportunistic fungal infections in plants.

Cross-continental emergence of Nannizziopsis barbatae disease may threaten wild Australian lizards.

Members of the genus Nannizziopsis are emerging fungal pathogens of reptiles that have been documented as the cause of fatal mycoses in a wide range of reptiles in captivity. Cases of severe, proliferative dermatitis, debility and death have been detected in multiple free-living lizard species from locations across Australia, including a substantial outbreak among Eastern water dragons (Intellagama lesueurii) in Brisbane, Queensland. We investigated this disease in a subset of severely affected lizards and identified a clinically consistent syndrome characterized by hyperkeratosis, epidermal hyperplasia, dermal inflammation, necrosis, ulceration, and emaciation. Using a novel fungal isolation method, histopathology, and molecular techniques, we identified the etiologic agent as Nannizziopsis barbatae, a species reported only once previously from captive lizards in Australia. Here we report severe dermatomycosis caused by N. barbatae in five species of Australian lizard, representing the first cases of Nannizziopsis infection among free-living reptiles, globally. Further, we evaluate key pathogen and host characteristics that indicate N. barbatae-associated dermatomycosis may pose a concerning threat to Australian lizards.

Application of Bacteriophages to Control Vibrio alginolyticus Contamination in Oyster (Saccostrea glomerata) Larvae.

Mortalities of bivalve larvae and spat linked with Vibrio spp. infection have been described in hatcheries since 1959, causing potential development of resistant bacteria. A reliable and sustainable solution to this problem is yet to be developed. Potential treatment of bacterial infection with bacteriophages is gaining interest in aquaculture as a more sustainable option for managing Vibrio spp. infection. This study assessed the effectiveness of bacteriophages (Φ-5, Φ-6, and Φ-7) against pathogenic Vibrio isolates (USC-26004 and USC-26005). These phage isolates were found to belong to the Myoviridae viral family. A total of 212 ORFs of Φ-5 were identified and annotated. The genome of this phage contained putative thymidine kinase and lysin enzyme. During infections with phages, the OD values of the isolates USC-26005 and USC-26004 remained stable at a much lower reading compared to the control after 9 h of incubation. Mortality rate of oyster (Saccostrea glomerata) larvae was 28.2 ± 3.5% in the bacteriophage treatment group, compared to 77.9 ± 9.1% in the bacterial treatment group after 24 h incubation. Findings of this study indicate that lytic phages might be utilized as potential bio-control agents of luminescent bacterial disease in oyster hatcheries.

Use of Bacteriophages to Control Vibrio Contamination of Microalgae Used as a Food Source for Oyster Larvae During Hatchery Culture.

Cultured microalgae are the primary food source for oyster larvae during hatchery culture and are a potential vector for Vibrio spp. infection of larval cultures. Bacteriophages have shown potential for controlling contamination of Vibrio spp. in aquaculture systems and their application could be an effective biological control method to eliminate such bacterial contamination of microalgae. This study investigated whether Vibrio-free microalgae sources could be ensured via the application of Vibrio specific phages. As a first step, four different Vibrio bacteriophages (belonging to the Myoviridae viral family) were isolated from marine waters in Queensland, Australia and used in challenge tests against a Vibrio host species, previously isolated from New South Wales oyster hatchery and found to be closely related to V. alginolyticus (ATCC 17749). The genome sequence of one of the four isolated bacteriophages, Vibrio Φ-2, that displayed strongest virulence against the host was determined. The 242446 bp genome of this bacteriophage was predicted to encode 217 proteins with an average GC content of 43.91%, containing putative thymidine kinases and a lysin enzyme. Application of these bacteriophages to pathogenic Vibrio spp. contaminating microalgae suspensions resulted in significant decreases in their numbers within 2 h. Findings indicated that direct application of bacteriophages to microalgae suspensions could be an effective method of reducing the occurrence of vibriosis in oyster hatcheries.

Comparative Genomics and Phenotypic Investigations Into Antibiotic, Heavy Metal, and Disinfectant Susceptibilities of Salmonella enterica Strains Isolated in Australia.

Salmonella enterica is recognized as a major contributor of gastrointestinal illness worldwide. Concerns have been raised over the increasing prevalence of antibiotic resistant strains of Salmonella isolated from animals and food, and the role of antibiotics and other antimicrobial agents such as biocides and heavy metals in the selection and dissemination of antibiotic resistant bacteria to human hosts. In this study the antibiotic, heavy metal and disinfectant resistance genotypes and phenotypes of 19 S. enterica isolates from food-producing animals were established using whole genome sequence analysis, disc diffusion, as well as broth or agar dilution methods. This study also investigated the genomic environment of resistance genes on mobile genetic elements and chromosomal DNA. An ampicillin and streptomycin resistant S. Infantis isolate in this study harbored a ß-lactamase (bla TEM-1 ), and two streptomycin resistance conferring genes (strA and strB) on a class 1 integron mobilized on a large conjugative plasmid. This plasmid also harbored two arsenic resistance gene cassettes. The arsenic resistance cassette, arsRCDAB, was also observed in two S. Singapore isolates with high tolerance to arsenate. A nalidixic acid resistant S. Typhimurium isolate was found to possess a mutation in gyrA resulting in amino acid change Asp87Gly and tetracycline resistant S. Typhimurium isolate was found to harbor efflux pump gene, tetA. No resistance (genotypic or phenotypic) was recorded to the disinfectants screened in this study. Taken together, results of this study showed a good correlation between predicted and measured resistances when comparing genotypic and phenotypic data, respectively. The findings of this study do not suggest resistance to clinically relevant antibiotics are widespread among Salmonella isolated from Australian food-producing animals.

Assessment of the Detrimental Impact of Polyvalent Streptophages Intended to be Used as Biological Control Agents on Beneficial Soil Streptoflora.

Streptophages are currently being investigated to control potato common scab, however, since a majority of streptophages are reported to be polyvalent, their potential to infect beneficial soil streptomycetes during the application process may have unintended consequences. To test this hypothesis, two phytopathogenic fungi, namely Fusarium solani and Rhizoctonia solani, were tested for their detrimental effect on the test crop wheat (Triticum aestivum cv. Gutha). F. solani caused a significant root weight reduction (34%) in the wheat plant and therefore was tested further in the pot trials with actinomycetes present. Sixty-seven streptomycete isolates from a Tasmanian potato farm were screened for their antifungal abilities against the two phytopathogenic fungi. Four actinomycetes found to be strongly antifungal were then tested for their disease-protective abilities against F. solani in pot trials again using wheat. Addition of the streptomycetes into the container media protected the plants against F. solani, indicating that streptomycetes have a disease-suppressive effect. A further pot trial was conducted to evaluate whether these beneficial streptomycete species would be affected by streptophage treatment and subsequently result in an increased risk of fungal infections. When streptophages were added to the pots, the shoot and root growth of wheat declined by 23.6% and 8.0%, respectively, in the pots with the pathogenic fungus compared to the control pots. These differences might suggest that removal of antifungal streptomycetes by polyvalent phages from plant rhizosphere when biocontrol of plant pathogenic streptomycetes (e.g. Streptomyces scabiei) is targeted might encourage secondary fungal infections in the farm environment. The presented data provide preliminary evidence that streptophage treatment of pathogenic streptomycetes may lead to an aggravated disease risk by soil-borne fungal pathogens when naturally present antagonists are removed. As a result, extensive farm site trials are required to determine the long-term detrimental impact of polyvalent streptophage treatments on beneficial soil streptoflora.

HSQC-TOCSY Fingerprinting-Directed Discovery of Antiplasmodial Polyketides from the Marine Ascidian-Derived Streptomyces sp. (USC-16018).

Chemical investigations on the fermentation extract obtained from an ascidian-derived Streptomyces sp. (USC-16018) yielded a new ansamycin polyketide, herbimycin G (1), as well as a known macrocyclic polyketide, elaiophylin (2), and four known diketopiperazines (3⁻6). The structures of the compounds were elucidated based on 1D/2D NMR and MS data. The absolute configuration of 1 was established by comparison of experimental and predicted electronic circular dichroism (ECD) data. Antiplasmodial activities were tested for the natural products against chloroquine sensitive (3D7) and chloroquine resistant (Dd2) Plasmodium falciparum strains; the two polyketides (1⁻2) demonstrated an inhibition of >75% against both parasite strains and while 2 was highly cytotoxic, herbimycin G (1) showed no cytotoxicity and good predicted water solubility.

HSQC-TOCSY Fingerprinting for Prioritization of Polyketide- and Peptide-Producing Microbial Isolates.

Microbial products are a promising source for drug leads as a result of their unique structural diversity. However, reisolation of already known natural products significantly hampers the discovery process, and it is therefore important to incorporate effective microbial isolate selection and dereplication protocols early in microbial natural product studies. We have developed a systematic approach for prioritization of microbial isolates for natural product discovery based on heteronuclear single-quantum correlation-total correlation spectroscopy (HSQC-TOCSY) nuclear magnetic resonance profiles in combination with antiplasmodial activity of extracts. The HSQC-TOCSY experiments allowed for unfractionated microbial extracts containing polyketide and peptidic natural products to be rapidly identified. Here, we highlight how this approach was used to prioritize extracts derived from a library of 119 ascidian-associated actinomycetes that possess a higher potential to produce bioactive polyketides and peptides.

Protective Effects of Bacteriophages against Aeromonas hydrophila Species Causing Motile Aeromonas Septicemia (MAS) in Striped Catfish.

To determine the effectivity of bacteriophages in controlling the mass mortality of striped catfish (Pangasianodonhypophthalmus) due to infections caused by Aeromonas spp. in Vietnamese fish farms, bacteriophages against pathogenic Aeromonashydrophila were isolated. A.hydrophila-phage 2 and A.hydrophila-phage 5 were successfully isolated from water samples from the Saigon River of Ho Chi Minh City, Vietnam. These phages, belonging to the Myoviridae family, were found to have broad activity spectra, even against the tested multiple-antibiotic-resistant Aeromonas isolates. The latent periods and burst size of phage 2 were 10 min and 213 PFU per infected host cell, respectively. The bacteriophages proved to be effective in inhibiting the growth of the Aeromonas spp. under laboratory conditions. Phage treatments applied to the pathogenic strains during infestation of catfish resulted in a significant improvement in the survival rates of the tested fishes, with up to 100% survival with MOI 100, compared to 18.3% survival observed in control experiments. These findings illustrate the potential for using phages as an effective bio-treatment method to control Motile Aeromonas Septicemia (MAS) in fish farms. This study provides further evidence towards the use of bacteriophages to effectively control disease in aquaculture operations.

Streptophage-mediated control of off-flavour taint producing streptomycetes isolated from barramundi ponds.

Off-flavour taint of aquaculture products is a global issue reducing consumer confidence in the farmed produce as they are taken up via the gills of fish, and deposited in the lipids of the animal. If the fish are not purged, resulting undesirable muddy earthy flavour taint can be tasted by consumers. These undesirable flavour and odour is caused by the terpenoid compounds namely geosmin and 2-methylisoborneol, produced as secondary metabolites by certain bacteria including the cyanobacteria and actinomycetes. Current strategies to remediate the problem rely on treating the symptoms not the cause and involve the use of time consuming purging methods and costly chemicals. Biological control using bacteriophages, specific to the problem causing bacteria, offers a natural alternative to chemical control, which might reduce further complications of copper based algaecides and its subsequent implications on water quality. In an adaptation of such biological control approach streptomycetes isolated from barramundi ponds were tested for their susceptibility to streptophages to understand whether host destruction via phage lysis would subsequently eliminate off-flavour taint productions by these isolates. Following the determination of the streptophage susceptibility of the isolates one of the most odourous streptomycete species (USC-14510) was selected to be tested further using different pond simulations resembling real-life applications. Geosmin was tested as the indicator of off-flavour taint production and as it has been previously reported that the cyanobacteria-actinomycete interactions occurring in ponds result in even greater levels of geosmin and 2-methylisoborneol, the geosmin levels for the isolate in the presence of cyanobacteria and streptophages were also tested. Findings indicated that the highly odourous Streptomyces species (USC-14510) once infected with streptophages, can lose its capacity to produce off-flavour taints. Pond simulation studies also revealed geosmin production was significantly reduced when streptophages were introduced into the pond water where streptomycete species were grown. The bacteriophage control method developed in the presented study might again confirm significant potential for the bacteriophage-mediated remediation strategy to be adapted by the aquaculture industry.

Isolation and characterization of Enterobacteriaceae species infesting post-harvest strawberries and their biological control using bacteriophages.

Strawberry is a significantly consumed fruit worldwide, mostly without being subjected to disinfection processes. During the harvest and transfer from farm to consumers as well as where organic farming practises have been employed, the surface of the fruit may become contaminated by pathogenic bacteria. Post-harvest strawberry fruits in punnets available for public consumption were thus screened for the presence of enteric bacteria in the Sunshine Coast region of Queensland, Australia. Some of the tested samples (13 %) were found to carry such bacteria and even in greater numbers if organic amendments were used (69 %). The bacteria were found to belong in the genera of Escherichia, Enterobacter, Raoultella, Klebsiella, Pantoea, Shigella, Citrobacter and Cronobacter within the family Enterobacteriaceae. Some of the isolates were found to adhere to Caco-2 cells representing human gut epithelium as well as carrying virulence and toxin genes. Resistance mostly against sulphafurazole, cefoxitin, ampicillin and nitrofurantoin was found among 14 different antimicrobial agents tested including 100 % resistance to cefoxitin and ampicillin in the genus Pantoea. In the second phase of the study, bacteriophages were isolated against the isolates and were subsequently applied to post-harvest fruits. A significant (P ≤ 0.001) reduction in the number of enteric bacteria was observed when a high-titre polyvalent bacteriophage suspension (×10(12) PFU/mL) was applied to the fruit surface. Bacteriophages also decreased the adhesion of the Escherichia coli isolates to Caco-2 cells. Findings might indicate that biological control using bacteriophages might be of significant value for the industry targeting to reduce pathogenic loads of bacteria on the fruit.