Targeting biotypes of Dactylopius tomentosus to improve effective biocontrol of Cylindropuntia spp. in Australia

Seven Dactylopius tomentosus (Lamarck) biotypes were collected from a range of Cylindropuntia spp. in Mexico, South Africa and United States of America (USA) and imported into quarantine facilities at the Ecosciences Precinct. Host range trials were conducted for each biotype and further assessed against the Cylindropuntia species that are naturalised in Australia to determine the most effective biotype for each species. Host range was confined to the Cylindropuntia for all seven biotypes. In the efficacy trials, C. imbricata (Haw.) F.M.Knuth was killed by the ‘imbricata’ biotype within 16 weeks and C. kleiniae (DC.) F.M.Knuth died within 26 weeks. Cylindropuntia fulgida var. mamillata (DC.) Backeb. and C. imbricata were killed by the ‘fulgida’ biotype within 18 weeks. On-going trials suggest that C. rosea (DC.) Backeb. could be controlled by either the ‘acanthocarpa’ or the ‘acanthocarpa × echinocarpa’ biotypes. Cylindropuntia spinosior (Englem.) F.M.Knuth was not susceptible to any of the D. tomentosus biotypes assessed. A clear designation of which D. tomentosus biotype is most suited for each Cylindropuntia species will improve and increase the effectiveness of biological control of these weed species

The host range of three biotypes of Dactylopius tomentosus (Lamarck) (Hemiptera: Dactylopiidae) and their potential as biological control agents of Cylindropuntia spp. (Cactaceae) in Australia

The host range of two newly imported biotypes of Dactylopius tomentosus and their potential as biological control agents of Cylindropuntia spp. were investigated. A third biotype (imbricata) of D. tomentosus previously released in Australia to control C. imbricata was also screened to determine if it will feed on other species of Cylindropuntia occurring in Australia. Efficacy trials were conducted to evaluate the ability of the biotypes to retard the growth or kill those plant species supporting development of four or more individuals in the host test trials. The host range of the three biotypes of D. tomentosus was restricted to the genus Cylindropuntia. However, the biotypes showed varying degrees of specificity within this genus. The imbricata biotype was the only biotype to develop on Australian C. rosea provenances, albeit with a range of developmental success on all C. rosea provenances tested. The Spanish provenance supported the highest development success followed by Grawin (NSW), Lorne Station (NSW) while the least preferred was the Mexican provenance. The rosea and cholla biotypes were unsuitable candidates to control C. rosea in Australia. However, the efficacy trials showed that the cholla biotype had a high impact on four of the eight naturalised Cylindropuntia species in Australia. This biotype established rapidly and the sustained feeding of one fecund female and her progeny killed potted plants of C. imbricata and C. fulgida at week 18. This biotype has the potential to be an effective agent against C. fulgida, C. imbricata, C. kleiniae and C. tunicata and, as a consequence, an application seeking its release in Australia has been lodged.

Otolith morphology of four mackerel species (Scomberomorus spp.) in Australia: Species differentiation and prediction for fisheries monitoring and assessment

Four species of large mackerels (Scomberomorus spp.) co-occur in the waters off northern Australia and are important to fisheries in the region. State fisheries agencies monitor these species for fisheries assessment; however, data inaccuracies may exist due to difficulties with identification of these closely related species, particularly when specimens are incomplete from fish processing. This study examined the efficacy of using otolith morphometrics to differentiate and predict among the four mackerel species off northeastern Australia. Seven otolith measurements and five shape indices were recorded from 555 mackerel specimens. Multivariate modelling including linear discriminant analysis (LDA) and support vector machines, successfully differentiated among the four species based on otolith morphometrics. Cross validation determined a predictive accuracy of at least 96% for both models. An optimum predictive model for the four mackerel species was an LDA model that included fork length, feret length, feret width, perimeter, area, roundness, form factor and rectangularity as explanatory variables. This analysis may improve the accuracy of fisheries monitoring, the estimates based on this monitoring (i.e. mortality rate) and the overall management of mackerel species in Australia.

Mode of Action of a Purified Antitumor Protein from the Proteinaceous Crystal of Bacillus thuringiensis subsp. thuringiensis on Yoshida Ascites Sarcoma Cells

A purified antitumor protein from the proteinaceous crystal of Bacillus thuringiensis subsp. thuringiensis inhibits the growth of Yoshida ascites sarcoma both in vivo and in vitro. Exogenous respiration of the tumor cells was unaffected by the protein at a concentration as high as 500 µg/ml. The antitumor protein inhibits the uptake and incorporation of labeled precursors into macromolecules. However, the ratio of incorporation over uptake is not affected by the protein. Further, the protein brings about the leakage of 260-nm-absorbing material, proteins, and 32P-labeled cellular constituents from the Yoshida ascites sarcoma cells. The results show that the action of the antitumor protein appears to alter the cellular permeability of the tumor cells.

Enhancement of immune response by the proteinaceous crystal of Bacillus thuringiensis Var thuringiensis

The proteinaceous crystal of Bacillus thuringiensis Var thuringiensis was found to enhance humoral immune response in rats and guinea pigs immunised with sheep red blood cells. The enhancement was due to the increased levels of both 19S and 7S antibodies in the sera of the treated animals. A novel synthesis of 7S haemolytic antibodies was observed in case of crystal treated animals.

Ecological impacts of Phlebiopsis gigantea biocontrol treatment against Heterobasidion spp. as revealed by fungal community profiling and population analyses

Wood decay fungi belonging to the species complex Heterobasidion annosum sensu lato are among the most common and economically important species causing root rot and stem decay in conifers of the northern temperate regions. New infections by these pathogens can be suppressed by tree stump treatments using chemical or biological control agents. In Finland, the corticiaceous fungus Phlebiopsis gigantea has been formulated into a commercial biocontrol agent called Rotstop (Verdera Ltd.). This thesis addresses the ecological impacts of Rotstop biocontrol treatment on the mycoflora of conifer stumps. Locally, fungal communities within Rotstop-treated and untreated stumps were analyzed using a novel method based on DGGE profiling of small subunit ribosomal DNA fragments amplified directly from wood samples. Population analyses for P. gigantea and H. annosum s.l. were conducted to evaluate possible risks associated with local and/or global distribution of the Rotstop strain. Based on molecular community profiling by DGGE, we detected a few individual wood-inhabiting fungal species (OTUs) that seemed to have suffered or benefited from the Rotstop biocontrol treatment. The DGGE analyses also revealed fungal diversity not retrieved by cultivation and some fungal sequence types untypical for decomposing conifer wood. However, statistical analysis of DGGE community profiles obtained from Rotstop-treated and untreated conifer stumps revealed that the Rotstop treatment had not caused a statistically significant reduction in the species diversity of wood-inhabiting fungi within our experimental forest plots. Locally, ISSR genotyping of cultured P. gigantea strains showed that the Rotstop biocontrol strain was capable of surviving up to six years within treated Norway spruce stumps, while in Scots pine stumps it was sooner replaced by successor fungal species. In addition, the spread of resident P. gigantea strains into Rotstop-treated forest stands seemed effective in preventing the formation of genetically monomorphic populations in the short run. On a global scale, we detected a considerable level of genetic differentiation between the interfertile European and North American populations of P. gigantea. These results strongly suggest that local biocontrol strains should be used in order to prevent global spread of P. gigantea and hybrid formation between geographically isolated populations. The population analysis for H. annosum s.l. revealed a collection of Chinese fungal strains that showed a high degree of laboratory fertility with three different allopatric H. annosum s.l. taxa. However, based on the molecular markers, the Chinese strains could be clearly affiliated with the H. parviporum taxonomical cluster, which thus appears to have a continuous distribution range from Europe through southern Siberia to northern China. Keywords: Rotstop, wood decay, DGGE, ISSR fingerprinting, ribosomal DNA

Pentitol phosphate dehydrogenases: Discovery, characterization and use in D-arabitol and xylitol production by metabolically engineered Bacillus subtilis

The ultimate goal of this study has been to construct metabolically engineered microbial strains capable of fermenting glucose into pentitols D-arabitol and, especially, xylitol. The path that was chosen to achieve this goal required discovery, isolation and sequencing of at least two pentitol phosphate dehydrogenases of different specificity, followed by cloning and expression of their genes and characterization of recombinant arabitol and xylitol phosphate dehydrogenases. An enzyme of a previously unknown specificity, D-arabitol phosphate dehydrogenase (APDH), was discovered in Enterococcus avium. The enzyme was purified to homogenity from E. avium strain ATCC 33665. SDS/PAGE revealed that the enzyme has a molecular mass of 41 ± 2 kDa, whereas a molecular mass of 160 ± 5 kDa was observed under non-denaturing conditions implying that the APDH may exist as a tetramer with identical subunits. Purified APDH was found to have narrow substrate specificity, converting only D-arabitol 1-phosphate and D-arabitol 5-phosphate into D-xylulose 5-phosphate and D-ribulose 5-phosphate, respectively, in the oxidative reaction. Both NAD+ and NADP+ were accepted as co-factors. Based on the partial protein sequences, the gene encoding APDH was cloned. Homology comparisons place APDH within the medium chain dehydrogenase family. Unlike most members of this family, APDH requires Mn2+ but no Zn2+ for enzymatic activity. The DNA sequence surrounding the gene suggests that it belongs to an operon that also contains several components of phosphotransferase system (PTS). The apparent role of the enzyme is to participate in arabitol catabolism via the arabitol phosphate route similar to the ribitol and xylitol catabolic routes described previously. Xylitol phosphate dehydrogenase (XPDH) was isolated from Lactobacillus rhamnosus strain ATCC 15820. The enzyme was partially sequenced. Amino acid sequences were used to isolate the gene encoding the enzyme. The homology comparisons of the deduced amino acid sequence of L. rhamnosus XPDH revealed several similar enzymes in genomes of various species of Gram-positive bacteria. Two enzymes of Clostridium difficile and an enzyme of Bacillus halodurans were cloned and their substrate specificities together with the substrate specificity of L. rhamnosus XPDH were compared. It was found that one of the XPDH enzymes of C. difficile and the XPDH of L. rhamnosus had the highest selectivity towards D-xylulose 5-phosphate. A known transketolase-deficient and D-ribose-producing mutant of Bacillus subtilis (ATCC 31094) was further modified by disrupting its rpi (D-ribose phosphate isomerase) gene to create D-ribulose- and D-xylulose-producing strain. Expression of APDH of E. avium and XPDH of L. rhamnosus and C. difficile in D-ribulose- and D-xylulose-producing strain of B. subtilis resulted in strains capable of converting D-glucose into D-arabitol and xylitol, respectively. The D-arabitol yield on D-glucose was 38 % (w/w). Xylitol production was accompanied by co-production of ribitol limiting xylitol yield to 23 %.

Role of Bacillus subtilis BacB in the Synthesis of Bacilysin

Bacilysin is a non-ribosomally synthesized dipeptide antibiotic that is active against a wide range of bacteria and some fungi. Synthesis of bacilysin (L-alanine-[2,3-epoxycyclohexano-4]-L-alanine) is achieved by proteins in the bac operon, also referred to as the bacABCDE (ywfBCDEF) gene cluster in B. subtilis. Extensive genetic analysis from several strains of B. subtilis suggests that the bacABC gene cluster encodes all the proteins that synthesize the epoxyhexanone ring of L-anticapsin. These data, however, were not consistent with the putative functional annotation for these proteins whereby BacA, a prephenate dehydratase along with a potential isomerase/guanylyl transferase, BacB and an oxidoreductase, BacC, could synthesize L-anticapsin. Here we demonstrate that BacA is a decarboxylase that acts on prephenate. Further, based on the biochemical characterization and the crystal structure of BacB, we show that BacB is an oxidase that catalyzes the synthesis of 2-oxo-3-(4-oxocyclohexa-2,5-dienyl)propanoic acid, a precursor to L-anticapsin. This protein is a bi-cupin, with two putative active sites each containing a bound metal ion. Additional electron density at the active site of the C-terminal domain of BacB could be interpreted as a bound phenylpyruvic acid. A significant decrease in the catalytic activity of a point variant of BacB with a mutation at the N-terminal domain suggests that the N-terminal cupin domain is involved in catalysis.

Nitric Oxide Is Involved in Mycobacterium bovis Bacillus Calmette-Guerin-Activated Jagged1 and Notch1 Signaling.

Pathogenic mycobacteria have evolved unique strategies to survive within the hostile environment of macrophages. Modulation of key signaling cascades by NO, generated by the host during infection, assumes critical importance in overall cell-fate decisions. We show that NO is a critical factor in Mycobacterium bovis bacillus Calmette-Guérin-mediated Notch1 activation, as the generation of activated Notch1 or expression of Notch1 target genes matrix metalloproteinase-9 (MMP-9) or Hes1 was abrogated in macrophages derived from inducible NO synthase (iNOS) knockout (iNOS(-/-)), but not from wild-type, mice. Interestingly, expression of the Notch1 ligand Jagged1 was compromised in M. bovis bacillus Calmette-Guérin-stimulated iNOS(-/-) macrophages, and loss of Jagged1 expression or Notch1 signaling could be rescued by NO donors. Signaling perturbations or genetic approaches implicated that robust expression of MMP-9 or Hes1 required synergy and cross talk between TLR2 and canonical Notch1-PI3K cascade. Further, CSL/RBP-Jk contributed to TLR2-mediated expression of MMP-9 or Hes1. Correlative evidence shows that, in a murine model for CNS tuberculosis, this mechanism operates in vivo only in brains derived from WT but not from iNOS(-/-) mice. Importantly, we demonstrate the activation of Notch1 signaling in vivo in granulomatous lesions in the brains of Mycobacterium tuberculosis-infected human patients with tuberculous meningitis. Current investigation identifies NO as a pathological link that modulates direct cooperation of TLR2 with Notch1-PI3K signaling or Jagged1 to regulate specific components of TLR2 responses. These findings provide new insights into mechanisms by which Notch1, TLR2, and NO signals are integrated in a cross talk that modulates a defined set of effector functions in macrophages.