No description available.

Phyla nodiflora var. minor y Alternanthera philoxeroides (lagunilla) son plantas invasoras consideradas malezas en Australia. Este país propuso el estudio de los enemigos naturales en el ambiente nativo, Argentina y Bolivia, con el fin de seleccionar posibles agentes de control biológico. Con este fin se llevaron a cabo viajes exploratorios para colectar plantas sintomáticas y aislar hongos patógenos. Asociados a Phyla nodiflora se encontraron Puccinia lantanae, Cercospora lippiae y Colletotrichum spp. En lo que se refiere a lagunilla los patógenos hallados fueron Uredo pacensis, Alternaria sp., Wilsoniana blitti y Colletotrichum spp. Se realizaron ensayos experimentales de inoculación, germinación, shock térmico, almacenamiento y velocidad de crecimiento con los patógenos hallados. Para P. nodiflora, Puccinia lantanae y C. lippiae podrían utilizarse como agentes de control clásico y las especies de Colletotrichum como agentes de control inundativo. Uredo pacensis y W. blitti podrían considerarse como agentes de control clásico de la forma terrestre de lagunilla una vez completados los estudios de especificidad. Se recomendaría el uso de Alternaria sp. y Colletotrichum spp. como micoherbicidas. Cuando se aclare la situación taxonómica de Phyla nodiflora en Australia y Argentina, se podrán definir con certeza los agentes de biocontrol de la variedad minor. / Phyla nodiflora var. minor and Alternanthera philoxeroides (alligator weed) are considered weeds in Australia. Studies of its natural enemies were realized in the native range, Argentina and Bolivia to select pathogenic fungi as biocontrol agents. Exploratory trips were carried out to collect diseased plants and to isolate pathogenic fungi. Puccinia lantanae, Cercospora lippiae and Colletotrichum spp. were found in P. nodiflora. Uredo pacensis, Alternaria sp., Wilsoniana blitti and Colletotrichum spp were found in alligator weed. Experimental assays (inoculation, germination, storaging, growth rate) were performed with the pathogens. For P. nodiflora, Puccinia lantanae and C. lippiae might be used as classical biocontrol agents and Colletotrichum species as inundative agents. Uredo pacensis and W. blitti would be considered as classical biocontrol agents after specificity is determined. Alternaria sp. and Colletotrichum spp. could be recommended as bioherbicides. Biocontrol agents of P. nodiflora var. minor will be thoroughly confirmed once taxonomic discrepancies between Argentina and Australia can be elucidated.

This work encompasses studies on the development of biocontrol strategies to manage the disease lettuce drop, caused by the fungi Sclerotinia sclerotiorum and S. minor, using the mycoparasitic fungus Coniothyrium minitans, and to better understand interactions during mycoparasitism at the biochemical level. Results from field experiments revealed that two applications of C. minitans at manufacturer recommended rates significantly reduced the incidence of lettuce drop caused by S. sclerotiorum but not by S. minor. Applications of other biocontrol products tested did not significantly reduce disease incidence caused by either pathogen. Sclerotium population studies revealed that soil populations of S. sclerotiorum in lettuce production fields ranged from 0.08 to 2.9 sclerotia/100g of soil and were generally aggregated in their distribution. Continued field studies revealed that there was no significant effect of irrigation (sprinkler vs furrow) on either the impact of sclerotium density or the efficacy of C. minitans. Studies on the evaluation of different application rates of Contans against S. minor revealed that two applications of Contans at 5 X manufactures recommended rates significantly reduced the disease incidence. Examination of sclerotial exudates of Sclerotinia spp. revealed that crude exudates from both Sclerotinia spp. stimulated C. minitans spore germination and the stimulation was due to compounds within the polar fraction. Studies on the role of lectin-carbohydrate binding during fungal-mycoparasite interactions revealed that many plant lectins as well as crude proteins extracted from sclerotia of either Sclerotinia spp. induced agglutination of C. minitans spores in vitro. Spore germination of C. minitans stimulated by sclerotial exudates of either Sclerotinia spp. was significantly inhibited in the presence of the lectin Con A but not other plant lectins. In vitro studies on the directional growth of C. minitans preceding mycoparasitism revealed the involvement of G proteins for optimal response of C. minitans toward Sclerotinia stimulus.

No description available.

No description available.

Economic losses caused by the two spotted spider mite (Tetranychus urticae Koch) in the agricultural industry prompted the initiation of this project to evaluate the potential of native and exotic Beauveria bassiana isolates as biocontrol agents against this pest. Before the selection of a potential mycopesticide for use at a commercial scale, several crucial criteria must be taken into consideration. The application of some of these criteria have been the focus of this study and include: production of virulent B. bassiana inoculum, an evaluation of the virulence of the fungus against T. urticae, monitoring of the fungal infection cycle in T. urticae and establishing a phylogenetic evaluation of B. bassiana isolates using rDNA sequence analysis. A two-stage in vitro diphasic fermentation process produced B. bassiana inoculum. In the first submerged phase the impact of nutrient treatments (carbon and nitrogen) at different concentrations (3% and 4%) in a 1:1 ratio was investigated for mycelium dry mass production and spore yield. The 4% nutrient concentration yielded a higher mycelium dry mass yield compared to the 3% and was therefore used in the second semi-solid phase to stimulate aerial conidia formation in response to low nutrient stress. The fungal structures produced during the first phase of the diphasic fermentation process were submerged conidia recognized as small, spherical structures with a smooth form. In comparison, inoculum of the second semi-solid phase produced aerial conidia with small, spherical, rough surfaces and a brittle appearance assumed to be related to nutrient deprivation. Nutritional parameters exploited in this study favoured conidia production for use as a potential mycopesticide. An in vitro bioassay compared the infectivity of exotic and native isolates of B. bassiana against T. urticae adults. All the isolates were pathogenic with mite mortality increasing over time. Differences in the virulence of the B. bassiana isolates were demonstrated suggesting host-specificity. With respect to the native isolates B. bassiana (PPRI 04305) was more virulent than the B. bassiana sensu latu isolates (PPRI 04304 and PPRI 04306). The differences in the virulence of the native isolates are reflections of genetic differences demonstrated in the phylogenetic analyses in this study. The results of the preliminary bioassay study suggest that B. bassiana has the potential as a biocontrol agent of T. urticae. Microscopy was used to morphologically visualise the post infection cycle of a native B. bassiana isolate (PPRI 04305) in the two-spotted spider mite. The infection cycle observed in the current study is in agreement with those described in a number of agricultural pests. However, aspects not observed before with T. urticae infection included limited hyphal growth on the cuticle surface before penetration, per os mode of entry, cuticular melanization, lateral hyphal development under the cuticle and aerial hyphal emergence through the setal annulum on the dorsal surface of the cadaver. These observations will stimulate further research in the development of B. bassiana as a mycoacaricide. rDNA analysis of the ITS1-5.8S-ITS2 regions of different Beauveria species was assessed for strain genotyping and population studies. Fitch parsimony and neighbour joining analyses displayed species differentiation and confirmed that B. bassiana was not a monophyletic group but a species complex. Distinct clades in the phylogenetic analyses in the current study were matched to four species of Beauveria: B. bassiana, B. cf. bassiana, Beauveria brongniartii and Beauveria caledonica species. Two South African isolates PPRI 04304 and PPRI 04306 morphologically assigned to B. bassiana, are assumed to be either B. caledonica or a close relative of B. caledonica based on the rDNA analysis. However, due to the lack of confirmation of the change of species identification of these native isolates, they are regarded as B. bassiana sensu latu. Results from this study demonstrated the importance of rDNA analysis in biocontrol studies for population studies and species differentiation. The material in this dissertation highlighted some important characteristics relevant for the biocontrol of T. urticae by B. bassiana. Aerial conidia produced by the cost-effective diphasic fermentation process were virulent against T. urticae and demonstrated high percentage mortalities. B. bassiana was shown to be a generalist pathogen with strain-dependent differences in nutrient preferences and virulence against the mite. Differences in the infectivity of the native isolates B. bassiana (PPRI 04305) and B. bassiana sensu latu (PPRI 04304 and PPRI 04306) were reflections of the genotypic separation of the isolates demonstrated by rDNA analysis. The results obtained from this research project are promising for the ongoing research and development of Beauveria isolates as efficient mycoacaricides against T. urticae for the South African agricultural market.

The purpose of this research was to explore the potential application of lytic phages to control Cronobacter spp. in infant formula. More than two hundred and fifty phages were isolated from various environmental samples against different strains of Cronobacter spp. Selected phages were characterized by morphology, host range, and cross infectivity. The genomes of five novel Cronobacter phages [vB_CsaM_GAP31 (GAP31), vB_CsaM_GAP32 (GAP32), vB_CsaP_GAP52 (GAP52), vB_CsaM_GAP161 (GAP161), vB_CsaP_GAP227 (GAP227)] were sequenced. Phage GAP32 possess the second largest phage genome sequenced to date, and it is proposed that GAP32 belongs to a new genus of “Gap32likeviruses”. Phages GAP52 and GAP227 are the first C. sakazakii podoviruses whose genomes have been sequenced. None of the sequenced genomes showed homology to virulent or lysogenic genes. In addition, in vivo administration of phage GAP161 in the hemolymph of Galleria mellonella larvae showed no negative effects on the wellbeing of the larvae and could effectively prevent Cronobacter infection in the larvae. A cocktail of five phages was highly effective for biocontrol of three Cronobacter sakazakii strains present as a mixed culture in both broth media and contaminated reconstituted infant formula. This phage cocktail could be potentially used to control C. sakazakii during preparation of infant formula but would first have to be clinically evaluated in mammalian models. / NSERC & DFO

The primary aim of this study was to investigate the effects of seed inoculation with the biocontrol agent Pseudomonas fluorescens strain F113 (producing the antibiotic DAPG) and its modified derivative strain F113G22 (with DAPG production disrupted) on the uptake of nitrogen by pea and wheat plants. Uptake of N by the two plant species was investigated in soil microcosms amended with 15N-labelled fertilisers (urea or ammonium nitrate) or 15N-labelled plant residues. Uptake of fertiliser-N was unaffected by inoculation. However, uptake of N derived from organic residues was enhanced in pea inoculated with either strain. In contrast, uptake of N by wheat was reduced in the presence of either strain F113 or F113G22, suggesting that the effects of these microbial inocula on N-mineralisation in the rhizosphere were dependent on plant species. It was subsequently found that microfaunal populations, especially soil nematodes in the rhizosphere of inoculated pea were significantly larger than those associated with the rhizosphere of non-inoculated controls. In wheat, however, microfaunal populations in the rhizosphere of inoculated plants were lower than those associated with noninoculated controls. These trends were repeated using simple sand microcosms into which soil bacteria and the bacterial-feeding nematode Caenorhabditis elegans were introduced. This suggested that effects on N-mineralisation were mediated by changes in populations of microbial-feeding microfauna. As a possible explanation for the increased nematode populations in the rhizosphere of inoculated pea plants, the nematicidal effects of pea seed exudates on C. elegans were investigated in small-scale sand systems. It was found that exposure to non-inoculated pea seeds reduced the short-term survival of C. elegans as compared to unamended sand, and that survival was greater in the presence of pea seeds inoculated with either strain than non-inoculated seeds, suggesting that nematicidal compounds released by germinating pea seeds were utilised by the P.fluorescens strains. No such effects were observed for wheat.

Antibiotic production kinetics of Streptomyces strain JS1 (an isolate selected from an industrial screen for biocontrol activity) and Streptomyces hygroscopicus (a culture collection strain with similar biocontrol activity to JS1) were examined. Growth-associated niphimycin production was observed in both strains during carbon-limited batch culture. Growth associated antibiotic production in carbon-limited medium has not been reported elsewhere and the antibiotic production physiology of biocontrol isolates has not been extensively studied in other laboratories. Increases in biomass and antibiotic production occurred simultaneously in JS1 (24h) and S. hygroscopicus (40h) carbon-limited cultures. Specific growth and antibiotic production rates peaked simultaneously (35h in S. hygroscopicus, 30h in JS1). Examination of the correlation between intracellular protein synthesis rate and niphimycin production rate was consistent with the relationship between these parameters proposed (in our laboratory and elsewhere) for the more frequently reported phenomenon of growth- dissociated antibiotic production. Evidence was obtained which resulted in a hypothesis that the unusual antibiotic production kinetics were a result of the unusually low affinity of JS1 for glucose. A novel approach (multi-compartment nonlinear modelling) to the determination of substrate affinity constants yielded a Ks value of 2.9mM which compares to 7.55muM (i.e. significantly higher affinity) value for Saccharopolyspora erythraea, a species which demonstrates the more common, growth dissociated form of production. Antibiotic production in nitrogen-limited culture was also growth- associated, but this has been reported elsewhere. Work reported here suggests that affinity for nitrogen substrate is significantly lower than that for glucose in S. erythraea (4.45mM compared to 7.55muM) a strain that also exhibits growth-associated antibiotic production under nitrogen limitation. This presumably explains the more growth-associated production kinetics observed in nitrogen-limited cultures. It is tempting to speculate a link between antibiotic production kinetics and biocontrol potential. A micro-organism capable of releasing anti-microbial product in synchrony with cell growth would presumably have more effect in reducing the rhizosphere microflora, prior to colonising the habitat, than a species producing the antibiotic as a secondary metabolite. If this hypothesis is justified, then it may explain the success of JS1 and S. hygroscopicus in the industrial screen. A mutant of JS1, unable to produce niphimycin, displayed diminished biocontrol capability, indicating that niphimycin has a role in the observed biological control effect, in this instance. An attempt to increase the biocontrol effectiveness of JS1 by enhancing niphimycin production in hydroponic and agar tomato culture systems was unsuccessful due to the production kinetics displayed by JS1. Manipulating culture conditions for increased niphimycin production inevitably resulted in increased JS1 growth which was associated with plant death. Electron microscopy suggested that this enhanced growth resulted in excessive colonisation of the root system, possibly resulting in plant death due to root oxygen starvation. The fungal pathogens Phytophthora capsici and Fusarium oxysporum, used as challenge organisms in the industrial screen, had significantly higher affinities for the substrates examined (15muM and < 10muM, respectively for glucose and 22muM and < 38muM, respectively, for nitrate) compared to the affinities of JS1 (2.9mM and 2.4mM, respectively, for glucose and nitrate) indicating that competition for nutrients was unlikely to account for the success of JS1 in the screen. An additional novel concept explored in this work was the use of a fractional factorial medium design procedure (the Plackett-Burman technique) for the attempted identification of nutrients that could be used to simultaneously enhance growth of biocontrol agents whilst inhibiting the growth of target pathogens. Nutritional requirements thus elucidated were compared to those of variant strains of S. hygroscopicus and other Streptomyces species.

Theory predicts that, upon release from natural enemies in the new range, invasive species will evolve to allocate more resources towards traits that improve their ability to compete with native species. The motivation for most biological control programs is to re-introduce native enemies in order to oppose this effect and reduce or even reverse some of the negative impacts of invasive species on native and economically important ecosystems. In many cases, multiple biocontrol agents are introduced, often under the assumption that their impacts will be complementary. However, studies that attempt to quantify the nature of interactions among biocontrol agents tend to find unique outcomes: as yet, no general patterns have emerged. Here I describe the findings of a study of the impacts of the introduction of one species of biocontrol agent of invasive purple loosestrife, the leaf beetle Neogalerucella almariensis, on the reproductive success of a second biocontrol agent, the flower-feeding weevil Nanophyes marmoratus. Somewhat surprisingly, I found that more adult weevils tended to emerge from plants that were simultaneously infested with both types of beetles. This was true even when differences in flower number among plants were taken into account. Moreover, there were more aborted flowers on an inflorescence where both biocontrol agents were present. Finally, more pollinators visited the plants that were infested by both biocontrol agents. The observational data also show a positive correlation between the presence of the leaf beetle larvae and the number of adult flower weevils found on an inflorescence. I discuss various explanations, including the possibility that biocontrol-induced changes in flowering phenology and plant nutritional compounds could be, at least partially, responsible for these findings.