Academic literature on the topic 'Genetic biocontrol'

ABSTRACT Mycoparasitic strains of Trichoderma are applied as commercial biofungicides for control of soilborne plant pathogens. Although the majority of commercial biofungicides are Trichoderma based, chemical pesticides, which are ecological and environmental hazards, still dominate the market. This is because biofungicides are not as effective or consistent as chemical fungicides. Efforts to improve these products have been limited by a lack of understanding of the genetic regulation of biocontrol activities. In this study, using gene knockout and complementation, we identified the VELVET protein Vel1 as a key regulator of biocontrol, as well as morphogenetic traits, in Trichoderma virens, a commercial biocontrol agent. Mutants with mutations in vel1 were defective in secondary metabolism (antibiosis), mycoparasitism, and biocontrol efficacy. In nutrient-rich media they also lacked two types of spores important for survival and development of formulation products: conidia (on agar) and chlamydospores (in liquid shake cultures). These findings provide an opportunity for genetic enhancement of biocontrol and industrial strains of Trichoderma, since Vel1 is very highly conserved across three Trichoderma species.

Entomopathogenic fungi play important roles in the control of populations of agricultural and disease vector pests in nature. The shortcomings of mycoinsecticides for pest management in the field cannot be completely overcome by improving single biocontrol properties of fungi. Therefore, enhancing the biocontrol potential of entomopathogenic fungi in multiple respects by genetic engineering is desirable. Transcription factors are usually involved in various important processes during fungal growth and pathogenesis via regulating a series of genes, and are important candidates for fungal improvement via genetic engineering. Herein, overexpression of MaSom1, a key transcription factor gene in the cAMP/PKA pathway, improves the biocontrol traits of Metarhizium acridum in multiple respects. When compared with WT, the MaSom1-overexpression strains exhibit enhanced tolerances to UV-B and heat shock, with increased mean 50% inhibition times by 66.9% and 155.2%, respectively. Advanced conidiation emerged accompanied by increased conidial yield up to 3.89 times after 3-day incubation for the MaSom1-overexpression strains compared to WT. Furthermore, when compared with WT, the virulence of the MaSom1-overexpression strains was also increased with the mean 50% lethality times reduced by 21.8% to 23.8%. Taken together, the MaSom1-overexpression improved the biocontrol potential of M. acridum in multiple respects. Our results provide insights into the application of key transcription factors for genetic engineering and offer a credible way to further improve the biocontrol potential of entomopathogenic fungi.

Breeding plants to improve the effectiveness of biocontrol agents is a promising approach to enhance disease suppression by microorganisms. Differences in biocontrol efficacy among cultivars suggest there is genetic variation for this trait within crop germplasm. The ability to quantify host differences in support of biological control is influenced by variation in host response to the pathogen and the dose of pathogen and biocontrol agent applied to the host. To assess the contribution of each of these factors to successful biocontrol interactions, we measured disease over a range of pathogen (Pythium) and biocontrol agent (Bacillus cereus UW85) inoculum doses. We fit dose-response models to these data and used model parameter estimates to quantify host differences in response to the pathogen and biocontrol agent. We first inoculated eight plant species separately with three species of Pythium and evaluated three dose-response models for their ability to describe the disease response to pathogen inoculum level. All three models fit well to at least some of the host-pathogen combinations; the hyperbolic saturation model provided the best overall fit. To quantify the host contribution to biological control, we next evaluated these models with data from a tomato assay, using six inbred tomato lines, P. torulosum, and UW85. The lowest dose of pathogen applied revealed the greatest differences in seedling mortality among the inbred lines, ranging from 40 to 80%. The negative exponential (NE) pathogen model gave the best fit to these pathogen data, and these differences corresponded to model parameter values, which quantify pathogen efficiency, of 0.023 and 0.091. At a high pathogen dose, we detected the greatest differences in biocontrol efficacy among the inbred lines, ranging from no effect to a 68% reduction in mortality. The NE pathogen model with a NE biocontrol component, the NE/NE biocontrol model, gave the best fit to these biocontrol data, and these reductions corresponded to model parameter values, which quantify biocontrol efficiency, of 0.00 and 0.038, respectively. There was no correlation between the host response to the pathogen and biocontrol agent for these inbred lines. This work demonstrates the utility of epidemiological modeling approaches for the study of biological control and lays the groundwork to employ manipulation of host genetics to improve biocontrol efficacy.

Pseudomonasputida strain PCL1760 is a biocontrol agent protecting plants from pathogens via the mechanism of competition for nutrients and niches (CNN). To confirm this mechanism as well as to adapt the strain for biotechnological applications, full genome analysis was compared with the known biotechnological model, P. putida S12, and other related species, which were analyzed on different genomic databases. Moreover, the antibacterial activity of PCL1760 was tested against Staphylococcus aureus, Pseudomonas aeruginosa, and Pseudomonas syringae. No genetic systems involved in antibiosis were revealed among the secondary metabolite clusters of the strain of PCL1760. The only antagonistic effect was observed against P. syringae, which might be because of siderophore (yellow-greenish fluorescence), although less than 19% pyoverdin biosynthesis clusters were predicted using the AntiSMASH server. P. putida PCL1760 in comparison with the Pseudomonas simiae strain PCL1751, another biocontrol agent acting solely via CNN, which lost its ‘luxury’ genes necessary for antibiosis or parasitism/predation mechanisms, but carries genetic systems providing motility. Interestingly, immunity genes (CRISPR/Cas and prophages) showed PCL1760 to be robust in comparison with S12, while annotation on OrthoVenn2 showed PCL1760 to be amenable for genetic manipulations. It is tempting to state that rhizobacteria using the mechanism of CNN are distinguishable from biocontrol agents acting via antibiosis or parasitism/predation at the genomic level. This confirms the CNN of PCL1760 as the sole mechanism for biocontrol and we suggest the strain as a new model for genetic engineering.

Aflatoxin is a carcinogenic mycotoxin produced by Aspergillus flavus. Non-aflatoxigenic (Non-tox) A. flavus isolates are deployed in corn fields as biocontrol because they substantially reduce aflatoxin contamination via direct replacement and additionally via direct contact or touch with toxigenic (Tox) isolates and secretion of inhibitory/degradative chemicals. To understand touch inhibition, HPLC analysis and RNA sequencing examined aflatoxin production and gene expression of Non-tox isolate 17 and Tox isolate 53 mono-cultures and during their interaction in co-culture. Aflatoxin production was reduced by 99.7% in 72 h co-cultures. Fewer than expected unique reads were assigned to Tox 53 during co-culture, indicating its growth and/or gene expression was inhibited in response to Non-tox 17. Predicted secreted proteins and genes involved in oxidation/reduction were enriched in Non-tox 17 and co-cultures compared to Tox 53. Five secondary metabolite (SM) gene clusters and kojic acid synthesis genes were upregulated in Non-tox 17 compared to Tox 53 and a few were further upregulated in co-cultures in response to touch. These results suggest Non-tox strains can inhibit growth and aflatoxin gene cluster expression in Tox strains through touch. Additionally, upregulation of other SM genes and redox genes during the biocontrol interaction demonstrates a potential role of inhibitory SMs and antioxidants as additional biocontrol mechanisms and deserves further exploration to improve biocontrol formulations.

Phenazine-1-carboxylic acid (PCA) produced by plant-beneficial Pseudomonas spp. is an antibiotic with antagonistic activities against Phytophthora infestans, the causal agent of potato late blight. In this study, a collection of 23 different PCA-producing Pseudomonas spp. was confronted with P. infestans in potato tuber bioassays to further understand the interaction existing between biocontrol activity and PCA production. Overall, the 23 strains exhibited different levels of biocontrol activity. In general, P. orientalis and P. yamanorum strains showed strong disease reduction, while P. synxantha strains could not effectively inhibit the pathogen’s growth. No correlation was found between the quantities of PCA produced and biocontrol activity, suggesting that PCA cannot alone explain P. infestans’ growth inhibition by phenazine-producing pseudomonads. Other genetic determinants potentially involved in the biocontrol of P. infestans were identified through genome mining in strains displaying strong biocontrol activity, including siderophores, cyclic lipopeptides and non-ribosomal peptide synthase and polyketide synthase hybrid clusters. This study represents a step forward towards better understanding the biocontrol mechanisms of phenazine-producing Pseudomonas spp. against potato late blight.

This research examines the impact of genetic diversity in dengue virus (DENV) on incubation period in vector mosquitoes, and on Wolbachia, a novel vector biocontrol agent. It also extends the DENV study to other flaviviruses (Zika and West Nile - Kunjin) and alphaviruses (Barmah Forest, Ross River and Sindbis) to provide evidence as to whether a heat-resistant strain of Wolbachia, wAlbB, can be used to suppress the transmission of these mosquito-borne viruses. The research outcomes show that viral genetic variation is reflected in transmission kinetics of DENV. Data from the Wolbachia experiments provide the groundwork for future in vivo mosquito trials.

Bibliography: leaves 207-220. Pseudomonas corrigata strain 2140 (Pc2140), isolated from wheat field soil in Australia, antagonises the take-all fungus, Gaeumannomyces graminis var. tritici (Ggt) in vitro and significantly reduces take-all symptoms on wheat in pot trials. This study investigates the mechanisms by which the biocontrol agent reduces the disease symptoms. Biochemical analysis of metabolites of P. corrugata 2140 reveal a number of compounds potentially antagonistic to Ggt and which may play a role in disease control. These include water-soluble antibiotics, siderophores, proteases, peptides and volatiles including hydrogen cyanide.

Thesis (MScAgric) -- University of Stellenbosch, 2002.
ENGLISH ABSTRACT: Fungi are an extremely diverse group of organisms and, by acting as pathogens, they can colonise various other organisms, including humans, plants and animals. The effect of this is usually detrimental, not only to agricultural crops and livestock, but also to human well-being. The extensive farming of crops and livestock requires persistent control of fungal populations, commonly through the use of chemical fungicides. However, the exclusive use of fungicides is no longer a sustainable practice, as a result of serious problems, such as increasing fungicide resistance in pathogen strains, the high costs of fungicides, as well as concern about the environment. The search by producers and scientists for alternative control measures is an ongoing process. The fungal cell wall consists of polysaccharides that not only playa role in protection of the fungi, but also in relaying signals for the invasion and infection of susceptible hosts. Chitin, a polysaccharide composed of N-acteylglucosamine (GleNAc) residues linked by P-1,4 glucosidic linkages, is one of the major components of the fungal cell wall, where it plays an important role in the apical growth of the vegetative hyphae. Chitinases (EC 3.2.1.14) are abundant proteins produced by a variety of microorganisms and plants and are necessary for the hydrolysis of the chitin polymer. During the invasion of many plant species by a pathogen, the production of a specific group of proteins, designated pathogenesis-related (PR) proteins that include chitinases, is induced as part of their defence response. Due to the facts that pathogenic fungi contain chitin in their cell walls and that plant chitinases are induced upon pathogen attack, chitinases have been confirmed as an integral and crucial part of the plant's natural defence response. Chitinases have increasingly been targeted to upregulate plants' endogenous disease resistance mechanisms through transgenic overexpression in a variety of hosts. Several species of fungi, including various Trichoderma spp., are potent biocontrol agents of plant pathogenic fungi and insects. The antagonistic activities of these biological control agents towards phytopathogens are based on the secretion of extracellular hydrolytic enzymes, such as cell wall-degrading chitinase enzymes. However, biological control is not restricted to naturally occurring biocontrol agents. Through the process of genetic transformation, other fungal or yeast species can be enhanced to produce their own chitinases or other antimicrobial substances more effectively in order to yield potent biocontrol agents. Various types of chitinases have been applied in the production of fungal resistant plants and some research has been done on the application of chitinases, from a variety of microorganisms, as biological control agents. In contrast, very little is known about the antifungal activity of the Saccharomyces cerevisiae chitinase enzyme, encoded by the CTS1-2 gene. The CTS1-2 gene was utilised in this study as a candidate for overexpression in both yeast and plant expression systems to analyse the ability of the encoding chitinase to inhibit fungal growth. The first objective of this study involved the high level expression and optimisation of the secretion of the CTS1-2 gene in S. cerevisiae to render recombinant yeast with enhanced antifungal abilities and with possible applications as a biocontrol agent to control plant pathogenic fungi. It was hypothesised that high-level expression and efficient secretion would be prerequisites in a biocontrol yeast strain. To this end, two strong promoters and terminators were included in the study and the secretion of the chitinase gene was evaluated by testing three different secretion signals. The secretion signals included: the native CTS1-2 secretion signal, the S. cerevisiae mating pheromone a-factor (MFa1) secretion signal, as well as the Trichoderma reesei f3-xylanase 2 (XYN2) secretion signal. The phosphoglycerate kinase 1 (PGK1) and alcohol dehydrogenase 2 (ADH2) promoters and terminators were employed to achieve high-level expression. The results obtained from the analysis of the recombinant yeasts showed that the PGK1 promoter-terminator constructs yielded high level CTS1-2-expressing and chitinase-producing strains of S. cerevisiae PRY488. The ability of the different secretion signals to efficiently secrete the overexpressed chitinase was analysed and it was found that the non-native secretion signals delivered significantly more protein to the extracellular environment. It was thus evident that the performance of the MFa1 and XYN2 secretion signals was superior to that of the native secretion signal. The antifungal activities of the recombinant chitinases produced by these constructs were tested in in vitro assays against Botrytis cinerea. The enzymes led to a significant reduction in hyphal development, caused by extreme structural damage to the hyphal tips, the hyphal cell walls as well as the ability of the fungus to form reproductive and survival structures, thereby confirming the antifungal abilities of this enzyme. The ADH2 promoter-terminator constructs yielded CTS1-2 transcripts, but no chitinase activity could be detected with any of these strains. The reasons for this still remain unclear. The second objective of this study was to assess the potential of the yeast chitinase gene to upregulate defence against fungal infection in planta. In order to elucidate this, the CTS1-2 gene was constitutively overexpressed in tobacco plants, targeting the chitinase both to the intra- and the extracellular environment. The results obtained showed that the transgenic tobacco lines regenerated in this study stably integrated the transgene, exhibiting transgene expression as well as the production of a biologically active yeast chitinase enzyme. The F, progeny were rigorously tested for resistance to B. cinerea, and both in vitro and in planta assays confirmed that the yeast chitinase increased the plant's tolerance to fungal infection; some of the lines showed disease resistance of 65 and 70%. The plants expressing an extracellularly targeted chitinase gene are still under evaluation. Interesting results are expected relating to the effect of the chitinase on the plant surface with regards to disease resistance to fungal pathogens. In conclusion, the combined set of results from both the yeast and plant overexpression studies has confirmed the strong antifungal effect of yeast chitinases. The yeast CTS1-2 chitinase could be instrumental in the development of a new generation of yeast strains with improved antifungal capabilities. This enzyme could also play an important role in genetic transformation technologies aimed at enhanced disease resistance.
AFRIKAANSE OPSOMMING: Swamme omsluit 'n uiterste diverse groep organismes wat mense, plante en diere deur patogeniese aksie kan koloniseer. Die uitkoms hiervan op landbougewasse, die veebedryf en menslike gesondheid is gewoonlik skadelik. Uitgebreide gewas- en veeboerderye benodig voortdurende beheer van fungiese populasies, tipies deur van chemiese swamdoders gebruik te maak. Die uitsluitlike gebruik van swamdoders is egter nie meer 'n lewensvatbare praktyk nie, hoofsaaklik as gevolg van probleme soos die opbou van weerstand van patogeniese rasse teen swamdoders, die hoë kostes van die middels, asook besorgheid oor die omgewing. Die soektog na alternatiewe beheermaatreëls deur produsente en wetenskaplikes bly 'n aaneenlopende proses. Die swamselwand bestaan uit polisakkariede wat nie net In rol in die beskerming van die swam speel nie, maar ook betrokke is in die oordrag van aanvals- en infeksieverwante seine in 'n vatbare gasheer. Chitien, 'n polisakkaried bestaande uit N-asetielglukosamien (GlcNAc) residu's gekoppel deur 13-1,4glukosidiese bindings, is een van die hoofkomponente van die swamselwand, waar dit 'n belangrike rol in die apikale groei van vegetatiewe hifes speel. Chitinases (EC 3.2.1.14) is proteïene wat oorvloedig deur 'n verskeidenheid van mikroërganismes en plante geproduseer word, waar hulle vir die hidrolise van die chitien polimeer noodsaaklik is. Tydens die infeksie van verskeie plantspesies deur In patogeen, word die produksie van 'n spesifieke groep proteïene, die sogenaamde patogeen-verwante (PR) proteïene wat chitinases insluit, as deel van die plant se verdedigingsreaksie geïnduseer. Die feit dat patogeniese swamselwande chitien bevat en dat plantchitinases tydens infeksie geïnduseer word, het daartoe gelei dat dit bevestig is dat chitinases In integrale en kritiese deel van die plant se natuurlike verdedigingsreaksie uitmaak. Chitinases word toenemend geteiken in pogings om die plant se intrinsieke siekteweerstandsmeganismes te verbeter deur transgeniese ooruitdrukking daarvan in 'n verskeidenheid van gashere. Verskeie swamspesies, insluitend verskillende Trichodenna-spesies, is kragtige bio-antagoniste van plantpatogeniese swamme. Die antagonistiese aksies van hierdie biologiese beheeragente teenoor fitopatogene is gebaseer op die uitskeiding van ekstrasellulêre hidrolitiese ensieme, soos die selwandverterende chitinase ensieme. Nietemin is biologiese beheer nie net tot bio-antagoniste wat natuurlik voorkom beperk nie. Deur die proses van genetiese transformasie kan ander swam- of gisspesies verbeter word om hul eie chitinases of ander antimikrobiese substanse meer effektief te produseer, wat aanleiding sal gee tot kragtige bio-antagoniste. Verskeie tipes chitinases is al in die produksie van swambestande plante ingespan en uitgebreide navorsing is gedoen op die toepassing van 'n reeks chitinases, afkomstig van 'n verskeidenheid van mikroërganismes, as biologiese beheeragente. In teenstelling is baie min bekend oor die antifungiese aktiwiteite van die Saccharomyces cerevisiae chitinase ensiem, wat deur die CTS1-2 geen ge-enkodeer word. Die CTS1-2-geen is in hierdie studie gebruik vir ooruitdrukking in beide gis- en plantuitdrukkingsisteme om die chitinase se vermoë om swamgroei te inhibeer, te ondersoek. Die eerste oorkoepelende oogmerk van hierdie studie het hoë-vlak uitdrukking en optimalisering van sekresie van die CTS1-2-geen in S. cerevisiae behels, met die toekomstige doelwit om 'n rekombinante gis met verbeterde antifungiese eienskappe en met moontlike toepassings as 'n bio-antagonis teen plantpatogeniese swamme te ontwikkel. Die hipotese was dat hoë-vlak uitdrukking en voldoende sekresie voorvereistes vir 'n bio-antagonisras is. Omdié rede is twee sterk promotors en termineerders by hierdie studie ingesluit en is die sekresie van die chitinase-geen geëvalueer deur drie verskillende sekresieseine te toets. Die sekresieseine sluit in: die wilde-tipe CTS1-2 sekresiesein, die S. cerevisiae paringsferomoon a-faktor (MFa1) sekresiesein, en die Trichoderma reesei p-xilanase (XYN2) sekresiesein. Die fosfogliseraat kinase 1 (PGK1) en alkohol dehidrogenase 2 (ADH2) promotors en termineerders is gebruik om hoë-vlak uitdrukking te dryf. Die resultate wat vanaf die analises van die rekombinante giste verkry is, het getoon dat die PGK1 promotor-termineerder konstrukte hoë-vlak CTS1-2-uitdrukkende en chitinase-produserende S. cerevisiae PRY488 rasse opgelewer het. Die vermoë van die verskillende sekresieseine om die ooruitgedrukte chitinase voldoende uit te skei, is geanaliseer, en daar is gevind dat die heteroloë sekresieseine aansienlik meer proteïene na die ekstrasellulêre omgewing geloods het. Dit was dus duidelik dat die MFa1 en XYN2 sekresieseine beter as die wilde-tipe sekresiesein presteer het. Die antifungiese aktiwiteit van die rekombinante chitinases wat deur hierdie konstrukte geproduseer is, is ook in in vitrotoetse teen Botryits cinerea getoets. Die teenwoordigheid van die ensieme het gelei tot 'n aansienlike afname in hife-ontwikkeling, veroorsaak deur ekstreme strukturele skade aan die hifepunte, die hifeselwande, asook die vermoë van die swam om voortplanting- en oorlewingstrukture te vorm. Die ADH2 promotor-termineerderkonstrukte het CTS1-2 transkripte vertoon, maar geen chitinase-aktiwiteite kon in hierdie konstrukte waargeneem word nie. Die redes hiervoor is tot op hede onbekend. Die tweede oogmerk van hierdie studie was om die potensiaal van die gischitinase om swaminfeksie in planta teë te werk, te ondersoek. Die CTS1-2-geen is konstitutief ooruitgedruk in tabakplante, waarin die chitinase na beide die intra- en ekstrasellulêre omgewing geteiken is. Resultate het getoon dat die geregenereerde transgeniese tabaklyne die transgeen stabiel geïntegreer het, transgeenuitdrukking vertoon en dat 'n biologies aktiewe chitinase-ensiem geproduseer is. 'n F1-generasie is aan strawwe toetse onderwerp om weerstand teen B. cinerea te ondersoek. Beide die in vitro en in planta toetse het bevestig dat die gischitinase die plant se verdraagsaamheid teenoor swaminfeksie verhoog het; sommige lyne het siekteweerstand van tussen 65 en 70% getoon. Die plante wat 'n ekstrasellulêre chitinase produseer, word steeds geëvalueer. Interessante resultate word verwag aangaande die effek van die chitinase op die plant se oppervlak met betrekking tot siekteweerstand teen swampatogene. Ten slotte, die gekombineerde stel resultate wat vanaf beide die gis- en plantuitdrukkingstudies verkry is, het die sterk antifungiese effek van gischitinases bevestig. Die gis CTS1-2 kan instrumenteel wees in die ontwikkeling van 'n nuwe generasie gisrasse met verbeterde antifungiese eienskappe. Die ensiem kan ook 'n belangrike rol in genetiese transformasietegnologieë, wat op verbeterde siekteweerstand gemik is, speel.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Rice blast caused by the fungus Magnaporthe oryzae B. Couch [anamorfo - Pyricularia oryzae Cav.] occurs in all rice growing regions of the world. The sustainable agriculture requires the introduction of biological control as one of the components in the integrated disease management. The microorganisms associated to plants are capable of producing secondary metabolites such as alkaloids which may have a role in biological control. The objective of the present study consists, isolation and identification of secondary metabolites of the micorrhizal fungus Rhizoctonia sp. obtained from Epidendrum nocturnum and evaluate in vitro and in vivo antagonism to M. oryzae. Ten fungal isolates were used to test the antibiosis against M. oryzae. The isolate En07 of Rhizoctonia sp. exhibited a greater halo of inhibition and consequently was considered the best in vitro antagonist to M. oryzae. Crude, mycelial and lyophilized extracts of micorrhizal isolate were obtained. The analysis by CCD of these three extracts showed positive results in relation to Dragendorff, indicating the presence of phenolics. The analysis of RMN 1H and masses showed the presence of aromatic hydrogens and phenolics. Five concentrations of each extract were prepared and utilized in the studies on in vitro mycelial inhibition of M. oryzae and observed 77.86% of pathogen reduction by crude extract (700 μg/mL). Two crude extract treatments (520 μg/ml and 120 μg/ml) significantly reduced the radial growth of the pathogen compared to control. The crude extract showed best results for mycelia inhibition of the pathogen, followed by lyophilized and mycelial extracts. In two trials, the crude extract at 0.52 μg.μL-1 also reduced the formation of appressoria of M. oryzae by 100%. Two greenhouse experiments were conducted on leaf blast suppression with the cultivar Primavera, using completely randomized design with three replications. In both these trials, the mycelial extract (1860 μg/ml and M.o 3x105) showed marked reduction of leaf blast severity in relation to control by 59.27% and 77.58% respectively. In the second trial, the second treatment (1040 μg/mL and M.o3x105) of crude extract reduced AUDPC by 64.63% compared to control. The results showed that the metabolites of Rhizoctonia sp. posses great potential for biological control of rice blast.
A brusone ocorre em todas as áreas produtoras de arroz do mundo, e é causada pelo fungo Magnaporthe oryzae B. Couch [anamorfo - Pyricularia oryzae Cav.]. Seu controle, realizado pelo manejo que integra resistência genética, práticas culturais e controle químico, requer a inserção de agentes biológicos, além de assegurar uma agricultura mais sustentável. Os microrganismos associados às plantas são capazes de produzir metabólitos secundários como os alcaloides que podem atuar no controle biológico. O presente trabalho objetivou isolar os metabólitos secundários do fungo micorrízico Rhizoctonia sp. obtido de Epidendrum nocturnum, e avaliar o antagonismo in vitro e in vivo com M. oryzae. Todos os ensaios foram conduzidos em delineamento inteiramente casualizado. Um ensaio de antagonismo foi avaliado, pareando-se, 10 isolados fúngicos e M. oryzae, e identificou-se que o isolado En07 foi o que apresentou o maior halo de inibição e consequentemente o melhor antagonismo in vitro sobre M. oryzae. Foram obtidos três extratos do isolado micorrízico (bruto, micelial e liofilizado) que foram analisados por quatro métodos diferentes e complementares. A análise por Cromatografia em Camada Delgada dos três extratos mostrou-se positiva frente ao reativo de Dragendorf, sugerindo a presença de compostos fenólicos. Na análise dos espectros de Ressonância Magnética Nuclear e de massas verificou-se a presença de hidrogênios aromáticos e de compostos fenólicos. Cinco concentrações de cada extrato foram preparadas e utilizadas nos ensaios de inibição micelial de M. oryzae in vitro e observou-se 77,86% de redução do patógeno pelo extrato bruto (700 μg/mL). Dois tratamentos do extrato bruto (520 μg/ml e 120 μg/ml) reduziram o crescimento radial do patógeno de forma significativa quando comparado com a testemunha. Verificou-se que o extrato bruto apresentou os melhores resultados para a inibição micelial do patógeno, seguido dos extratos liofilizado e do micélio. Em dois ensaios o extrato bruto a 0,52 μg.μL-1 também reduziu o número de apressórios formados de M. oryzae em 100%. Foram conduzidos dois ensaios de supressão de brusone foliar em arroz, in vivo, com a cultivar Primavera em três repetições. Nos dois ensaios destacou-se o extrato micelial (1860 μg/ml e M.o 3x105) que proporcionou maiores reduções da severidade de brusone nas folhas em relação ao controle com 59,27% e 64,63%, respectivamente. No segundo ensaio o tratamento 2 (1040 μg/mL e M.o3x105) do extrato bruto reduziu a AACPD em 24,93% em relação à testemunha. Os resultados mostraram que o metabólito de Rhizoctonia sp. possui grande potencial para o controle biológico da brusone.

The scale insect predator Chilocorus nigritus is considered as one of the most successful and important biological control agents in classical biocontrol. The ladybird is currently a commercial product, used for scale insect control in glasshouse environments, but despite widespread success in wild field releases, use of the predator in glasshouses has achieved only moderate success. This study aimed to find out which factors may affect the success of C. nigritus in glasshouse pest control. Two key factors with potential to have a dramatic impact on the predator were identified; genetic variability within and between insect populations, and male killing endosymbiotic bacteria, known to have a diverse range of effects on up to 70% of all insect species. Beetle strains were sourced from several insectaries and geographical locations. DNA sequencing determined significant genetic differences between biotypes of C. nigritus from different localities, indicating that C. nigritus exists as a series of functional biotypes across its range. All biotypes were tested for Wolbachia, Rickettsia and Spiroplasma infections, identifying Rickettsia and Wolbachia presence in most populations. Uninfected sub-lines of these strains were therefore created via tetracycline treatment. A number of bionomic characteristics of the beetle were compared across these strains and infection types in order to assess the influence of genetics and bacterial endosymbionts. Significant effects of endosymbionts were noted in fecundity and prey consumption, and genetically distinct biotypes varied in their prey consumption. A combination of biotypes and infection types also provided successful suppression of scale insects in glasshouse trials at Royal Botanical Gardens, Kew, and beetles were shown to produce a defence pheromone similar to hippodamine, which could potentially be another strong influence on the species’ ecology. This thesis suggests that variability within a predator population may be an asset, rather than a hindrance.

Les nématodes à kyste sont d’importants parasites des cultures, causant des pertes économiques élevées. Chez ces nématodes, l'éclosion est stimulée par les exsudats radiculaires libérés par la plante hôte. Le retrait des derniers nématicides nécessite le développement de solutions alternatives pour protéger les cultures. Ainsi, l’utilisation d’exsudats radiculaires pour provoquer une « éclosion suicide » des nématodes en l’absence de plantes hôtes représente un intérêt dans le management de ces parasites. Cette thèse visait à anticiper l'efficacité de cette stratégie, en prenant compte la diversité génétique côté nématodes et la composition microbiotique des sols. Tout d’abord, le niveau de dépendance entre les exsudats radiculaires d'espèces sauvages de Solanum et les populations de Globodera pallida sur le trait de vie éclosion a étéévalué et a mis en évidence un effet fort de la provenance géographique des exsudats. Ensuite les exsudats radiculaires de différentes espèces végétales ont été testés sur l'éclosion de populations représentatives de la diversité génétique de trois nématodes à kyste. Malgré des différences significatives obtenues entre les populations pour une espèce de nématode donnée, les exsudats radiculaires permettent une éclosion importante des nématodes. Enfin, l'impact des communautés microbiennes du sol sur l'efficacité des exsudats radiculaires a été mesuré. Des différences significatives d'éclosion suicide entre différents sols ont été obtenues, mais avec un taux d'éclosion élevé. Ces travaux fournissent des éléments clés pour l’utilisation d’exsu
Cyst nematodes are among the most harmful pests of cultivated crops causing important economic losses. For cyst nematodes, the hatching is stimulated by root exudates released by the host plant. The removal of chemical nematicides requires development of alternative approaches to protect crops. For this purpose, root exudates may constitute an effective and innovative biocontrol method that could be used in the absence of the host plant to induce a “suicide hatching” of nematode and to control cyst nematodes’ pressure on crop. This work aimed to anticipate the effectiveness of the suicide hatching strategy, taking into account the influence of the genetic diversity of nematode populations and the microbial composition of soils. First, the level of dependence between root exudates from wildSolanum species and Globodera pallida populations for the hatching trait was evaluated and highlighted a strong effect of the geographical location of root exudates. Second, root exudates from different plant species were tested on the hatching of representative populations of the genetic diversity for three cyst nematodes. Significant differences were obtained among populations for a given nematode species but root exudates provided a high level of hatching of nematodes. Third, the impact of microbial communities of soil on the efficiency of root exudates to stimulate the hatching was measured. Significant differences of suicide hatching between different soils were obtained but the hatching rate remained high. These research efforts provide key elements for the development of root exud

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
The tomato is a solanaceous plant who became quite prominent in the last century, not only by the pleasant taste, but also for being a rich source of amino acids, organic acids, vitamins, as well as the high concentration of beneficial substances, including lycopene. To meet the growing demand for tomatoes, various breeding programs were executed, aiming an increment in production, productivity and fruit quality, such as the reduction of secondary metabolites. By proceeding crossings to improve agronomic traits of the culture, genes conferring hardiness and hence resistance to disease were probably lost, favoring the increased attack of pests and diseases. Among the most troubling diseases stands the vascular wilt caused by Fusarium oxysporum f. spp. lycopersici. The difficulty of controlling this disease comes from the intrinsic characteristics of the pathogen, such as high adaptability to the underground environment while associated with the host and the production of resistance structures that remain viable in the soil for a long time. Genetic resistance stands out as the main tool in the control of this pathogen, which requires a continuous accession characterization program and the identification of sources of resistance, ensuring the introgression of 'I' genes that express resistance to physiologic races of the pathogen. Coupled with genetic resistance, the use of microorganisms as antagonists and their introduction in the pathogen´s microhabitat has become a promising alternative in the context of the management of root diseases of tomato, highlighting the colonizing bacteria from the rhizosphere of plants, termed rhizobacteria, as the main biocontrol agents. This study aimed to identify resistant tomato accessions to three physiological races of Fusarium oxysporum f. spp. lycopersici, by morphological and molecular methods, as well as detect multiple sources of resistance from the germplasm bank of EMBRAPA CNPH. The assay consisted of an evaluation of 28 varieties, 3 hybrids, 32 strains and 19 wild accessions, totaling 82 accessions. The susceptibility or resistance was confirmed by visualization of DNA bands on an agarose gel generated by specific molecular markers capable of amplifying regions of the target genes I-1, I-2 and I-3, expressing resistance to classes 1, 2 and 3 of F. oxysporum f. spp. lycopersici, respectively. To enhance the durability of this resistance, 10 rhizobacteria of the Bacillus genus were tested as biocontrol agents of wilt in greenhouse assays. In parallel, in vitro root colonization, antibiosis and detection of potential biocontrol genes involved in the tests were conducted. All Bacillus isolates showed variable levels of control of the pathogen in vitro, while the UFG-07 (Bacillus subtilis) and UFG-10 (Bacillus circulans) isolates have excelled in disease suppression in the greenhouse, which reinforces the hypothesis of substances acting as an antimicrobial control.
O tomateiro é uma solanácea que se tornou bastante proeminente no século passado, não só pelo agradável paladar, mas também por ser uma rica fonte de aminoácidos, ácidos orgânicos, vitaminas, como também pela grande concentração de substâncias benéficas, entre elas o licopeno. Para atender à crescente demanda pelo tomate, vários programas de melhoramento visando o aumento da produção, produtividade, qualidade dos frutos, como a diminuição de metabólitos secundários, foram empreendidas. Ao se procederem os cruzamentos visando melhorar as características agronômicas da cultura, genes que conferiam rusticidade e, consequentemente, resistência às doenças, foram provavelmente perdidos, favorecendo o aumento do ataque de pragas e doenças na cultura do tomateiro. Dentre as doenças mais preocupantes destaca-se a murcha vascular causada pelo fungo Fusarium oxysporum f. sp. lycopersici. A dificuldade de controle desta doença advém de características intrínsecas do patógeno, como alta adaptabilidade ao ambiente subterrâneo em associação com o hospedeiro e pela produção de estruturas de resistência que ficam viáveis no solo por um longo tempo. A resistência genética destaca-se como a principal ferramenta no controle deste fitopatógeno, o que requer um programa contínuo de caracterização de acessos e de identificação de fontes de resistência, garantindo a introgressão de genes ‘I’ que expressam resistência as raças fisiológicas do patógeno. Aliado a resistência genética, o uso de microrganismos como agentes antagonistas e sua introdução no microhabitat do patógeno, tem-se tornado como alternativa promissora no contexto do manejo de doenças radiculares do tomateiro, com destaque para as bactérias colonizadoras da rizosfera de plantas, denominadas rizobacterias, como os principais agentes de biocontrole. O objetivo deste trabalho visou identificar acessos de tomateiro resistentes às 3 raças fisiológicas de Fusarium oxysporum f. sp. lycopersici, pelos métodos morfológico e molecular, assim como detectar fontes de resistência múltipla no banco de germoplasma da EMBRAPA CNPH. O ensaio consistiu na avaliação de 28 variedades, 3 híbridos, 32 linhagens e 19 acessos selvagens, totalizando 82 acessos. A resistência ou suscetibilidade foi corroborada pela visualização de bandas de DNA em gel de agarose, ao utilizar-se de marcadores moleculares específicos para amplificar das regiões alvo, gene I-1, I-2 e I-3 que expressam resistência à raça 1, 2 e 3 de Fusarium oxysporum f. sp. lycopersici, respectivamente. Visando maior durabilidade da resistência, 10 rizobacterias do gênero Bacillus foram testadas como agentes de biocontrole da murcha do tomateiro, em casa de vegetação. Paralelamente foram conduzidos, in vitro, antibiose e detecção de possíveis genes envolvidos no biocontrole. Isolados de Bacillus apresentaram controle variável do patógeno in vitro, enquanto que os isolados UFG-07 (Bacillus subtilis) e UFG-10 (Bacillus circulans) se destacaram na supressão da doença em casa de vegetação.

Valdensinia heterodoxa Peyronel is an ascomycete fungus currently being considered as a potential biocontrol agent for Gaultheria shallon Pursh. (salal). In order to design an effective biocontrol agent and to assess its effectiveness and risks, the population structure of both V. heterodoxa and salal must be investigated. Infected salal leaves were collected from three geographically separate populations on Vancouver Island and coastal mainland British Columbia. Uninfected salal was collected from two additional sites. V. heterodoxa was cultured from the infected leaves and single spore cultures were obtained prior to DNA isolation. Amplified fragment length polymorphisms (AFLPs) were used to generate individual DNA fingerprints for each isolate. Of the 214 loci analyzed, 30 % were polymorphic, suggesting low genetic diversity. There were many shared haplotypes within each population, and as expected, analysis of pairwise kinship coefficients showed that as spatial distance increased, genetic similarity decreased. Analysis of molecular variance (AMOVA) between populations revealed significant genetic differentiation between populations with an FST of 0.18, perhaps a result of limited gene flow. Salal DNA was isolated from leaf tissue and AFLPs were used to fingerprint individuals resulting in 230 loci, which were 89.7% polymorphic on average. Within population diversity was high, with an average observed heterozygosity of 0.49. In addition, due to the high ploidy level of salal (octoploid), the results obtained from the dominant AFLP markers likely underestimate the actual genetic diversity in the populations. Populations were poorly differentiated (FST 0.096 ) , suggesting high gene flow among populations. Within one population (Shawnigan Lake), genetic similarity decreased with increased geographic distance and showed little evidence of clonal population structure. ii When the genetic variation in V. heterodoxa was compared to that in salal, high correlations of alleles observed between the species suggest that different V. heterodoxa pathogenicity groups, or salal varieties with varying levels of susceptibility, could be contributing to the distribution of V. heterodoxa in these populations. Overall, the findings from the genetic analyses were used to discuss the potential risks of using V. heterodoxa as a biocontrol agent for salal and suggest that with low diversity and high population differentiation, the effectiveness of V. heterodoxa as a biocontrol may be limited to use within local salal populations or in combination with other control methods to effectively manage salal in forested areas.

Bibliography: leaves 207-220.
220 leaves : ill. ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Pseudomonas corrigata strain 2140 (Pc2140), isolated from wheat field soil in Australia, antagonises the take-all fungus, Gaeumannomyces graminis var. tritici (Ggt) in vitro and significantly reduces take-all symptoms on wheat in pot trials. This study investigates the mechanisms by which the biocontrol agent reduces the disease symptoms. Biochemical analysis of metabolites of P. corrugata 2140 reveal a number of compounds potentially antagonistic to Ggt and which may play a role in disease control. These include water-soluble antibiotics, siderophores, proteases, peptides and volatiles including hydrogen cyanide.
Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 1996

Five Pseudomonas syringae pv. syringae bacteriophages were isolated from the quince, apple and pear. After a detailed study, the isolated bacteriophages could be used for biocontrol of the bacterial canker patho-gen. One of the isolated phages was active against the causative agents of bacterial canker and fire blight.

Enhancing the activity of biocontrol agents could be the most important factor in their success in controlling fruit disease and their ultimate acceptance in commercial disease management. Direct manipulation of a biocontrol agent resulting in enhancement of diseases control could be achieved by using recent advances in molecular biology techniques. The objectives of this project were to isolate genes from yeast species that were used as postharvest biocontrol agents against postharvest diseases and to determine their role in biocontrol efficacy. The emphasis was to be placed on the yeast, Candida oleophila, which was jointly discovered and developed in our laboratories, and commercialized as the product, Aspire. The general plan was to develop a transformation system for C . oleophila and either knockout or overexpress particular genes of interest. Additionally, biochemical characterization of the lytic peptides was conducted in the wild-type and transgenic isolates. In addition to developing a better understanding of the mode of action of the yeast biocontrol agents, it was also our intent to demonstrate the feasibility of enhancing biocontrol activity via genetic enhancement of yeast with genes known to code for proteins with antimicrobial activity. Major achievements are: 1) Characterization of extracellular lytic enzymes produced by the yeast biocontrol agent Candida oleophila; 2) Development of a transformation system for Candida oleophila; 3) Cloning and analysis of C.oleophila glucanase gene; 4) Overexpression of and knockout of C. oleophila glucanase gene and evaluating its role in the biocontrol activity of C. oleophila; 5) Characterization of defensin gene and its expression in the yeast Pichiapastoris; 6) Cloning and Analysis of Chitinase and Adhesin Genes; 7) Characterization of the rnase secreted by C . oleophila and its inhibitory activity against P. digitatum. This project has resulted in information that enhanced our understanding of the mode of action of the yeast C . oleophila. This was important step towards enhancing the biocontrol activity of the yeast. Fungal cell wall enzymes produced by the yeast antagonist were characterized. Different substrates were identified to enhance there production in vitro. Exo-b-1, 3 glucanase, chitinase and protease production was stimulated by the presence of cell-wall fragments of Penicillium digitatum in the growing medium, in addition to glucose. A transformation system developed was used to study the role of lytic enzymes in the biocontrol activity of the yeast antagonist and was essential for genetic manipulation of C . oleqphila. After cloning and characterization of the exo-glucanase gene from the yeast, the transformation system was efficiently used to study the role of the enzyme in the biocontrol activity by over-expressing or knocking out the activity of the enzyme. At the last phase of the research (still ongoing) the transformation system is being used to study the role of chitinase gene in the mode of action. Knockout and over expression experiments are underway.

One of the emerging technologies is the use of microbial agents for the control of postharvest diseases of fruits and vegetables. A number of antagonistic microorganisms have been discovered which have the potential to effectively control postharvest diseases. Some of this technology has been patented and commercial products such as AspireTM (Ecogen Corporatin, Langhorne, PA, USA), Biosave 10TM and Biosave 11TM (Ecoscience Inc., Worchester, MA, USA) have been registered for commercial use. The principal investigator of this project was involved in developing the yeast-based biofungicide-AspireTM and testing its efficacy under commercial conditions. This research project was initiated to fill the gap between the knowledge available on development and commercial implementation of yeast biocontrol agents and basic understanding of various aspects related to introducing yeast antagonists to fruit surfaces, along with verification of population genetics. The main objectives of this study were: Study ecology, population dynamics and genetic diversity of the yeast antagonists Candida guilliermondii, C. oleophila, and Debaryomyces hansenii, and study the effect of preharvest application of the yeast antagonist C. oleophila naturally occurring epiphytic microbial population and on the development of postharvest diseases of citrus fruit during storage. Our findings, which were detailed in several publications, have shown that an epiphytic yeast population of grapefruit able to grow under high osmotic conditions and a wide range of temperatures was isolated and characterized for its biocontrol activity against green mold decay caused by Penicillium digitatum. Techniques based on random amplified polymorphic DNA (RAPD) and arbitrary primed polymerase chain reaction (ap-PCR), as well as homologies between sequences of the rDNA internal transcribed spacers (ITS) and 5.8S gene, were used to characterize the composition of the yeast population and to determine the genetic relationship among predominant yeast species. Epiphytic yeasts exhibiting the highest biocontrol activity against P. digitatum on grapefruit were identified as Candida guilliermondii, C. oleophila, C. sake, and Debaryomyces hansenii, while C. guilliermondii was the most predominant species. RAPD and ap-PCR analysis of the osmotolerant yeast population showed two different, major groups. The sequences of the ITS regions and the 5.8S gene of the yeast isolates, previously identified as belonging to different species, were found to be identical. Following the need to develop a genetically marked strain of the yeast C. oleophila, to be used in population dynamics studies, a transformation system for the yeast was developed. Histidine auxotrophy of C. oloephila produced using ethyl methanesulfonate were transformed with plasmids containing HIS3, HIS4 and HIS5 genes from Saccharomyces cerevisiae. In one mutant histidin auxotrophy was complemented by the HIS5 gene of S. cerevisiae is functionally homologous to the HIS5 gene in V. oleophila. Southern blot analysis showed that the plasmid containing the S. cerevisiae HIS5 gene was integrated at a different location every C. oleophila HIS+ transformant. There were no detectable physiological differences between C. oleophila strain I-182 and the transformants. The biological control ability of C. oleophila was not affected by the transformation. A genetically marked (with b-glucuronidase gene) transformant of C. oleophila colonized wounds on orange fruits and its population increased under field conditions. Effect of preharvest application of the yeast C. oleophila on population dynamics of epiphytic microbial population on wounded and unwounded grapefruit surface in the orchard and after harvest was also studied. In addition, the effect of preharvest application of the yeast C. oleophila on the development of postharvest decay was evaluated. Population studies conducted in the orchard showed that in control, non-treated fruit, colonization of wounded and unwounded grapefruit surface by naturally occurring filamentous fungi did not vary throughout the incubation period on the tree. On the other hand, colonization of intact and wounded fruit surface by naturally occurring yeasts was different. Yeasts colonized wounded surface rapidly and increased in numbers to about two orders of magnitude as compared to unwounded surface. On fruit treated with the yeast and kept on the tree, a different picture of fungal and yeast population had emerged. The detected fungal population on the yeast-treated intact surface was dramatically reduced and in treated wounds no fungi was detected. Yeast population on intact surface was relatively high immediately after the application of AspireTM and decreased to than 70% of that detected initially. In wounds, yeast population increased from 2.5 x 104 to about 4x106 after 72 hours of incubation at 20oC. Results of tests conducted to evaluate the effect of preharvest application of AspireTM on the development of postharvest decay indicated the validity of the approach.

The objectives of the research in this proposal were to (A) identify synergy among proteins that provide enhanced activity over single proteins for control of plant pathogenic fungi, (B) clone and characterize genetic sequences coding for proteins with ability to control pathogenic fungi, (C) produce transgenic organisms with enhanced biocontrol ability using genes and gene combinations and determine their efficiency in protecting plants against plant pathogenic fungi. A related objective was to produce disease-resistant plants. Fungal cell wall degrading enzymes from any source are strongly synergistic with any membrane active compound and, further, different classes of cell wall degrading enzymes are also strongly synergistic. We have cloned and sequenced a number of genes from bacterial and fungal sources including five that are structurally unrelated. We have prepared transgenic fungi that are deficient in production of enzymes and useful in mechanistic studies. Others are hyperproducers of specific enzymes that permit us, for the first time, to produce enzymes from T. harzianum in sufficient quantity to conduct tests of their potential use in commercial agriculture. Finally, genes from these studies have been inserted into several species of crop plants were they produce a high level of resistance to several plant pathogenic fungi.

The overall goal of this research was to elucidate the factors affecting early development of Metarhizium spp. (previously named M. anisopliae) on ticks or tick cuticle extracts and the molecular basis of these early infection processes. The original objectives were: 1. Characterize the pre-penetration events (adhesion, germination and appressorium formation) of spores of M. anisopliae strains with high or low virulence during tick infection. 2. Create GFP-expressing strains of M. anisopliae tick pathogens having high and low virulence to compare their progress of infection by microscopy. 3. Use microarray analyses, primarily with existing M. anisopliae EST sequences in GenBank, to identify and characterize fungal genes whose expression is regulated in response to host cuticle extracts. Objective 3 was later modified (as approved by BARD) to use RNAseq to characterize the early stages of fungal gene expression during infection of intact host cuticles. This new method provides a massively larger and more informative dataset and allows us to take advantage of a) recently published genomes of Metarhizium robertsii and M. acridum for RNAseq data analysis, and b) newly developed and highly efficient cDNA sequencing technologies that are relatively low cost and, therefore, allow deep sequencing of multiple transcriptome samples. We examined pre-penetration and penetration events that differentiate high and low virulence strains of Metarhizium spp., focusing on spore adhesion, germination, appressorium formation, and penetration of tick integuments. Initiation of fungal infection was compared on susceptible and resistant tick species at different tick developmental stages. In vitro studies comparing the effects of protein and fatty acid profiles from tick cuticle extracts demonstrated that resistant tick cuticles contain higher concentrations of specific lipids that inhibit fungal development than do susceptible tick cuticles, suggesting one mechanism of Ixodidae resistance to fungal entomopathogens (Objective 1). We used molecular markers to determine that the three M. anisopliae strains from Israel that we studied actually were three distinct species. M. brunneum is highly virulent against the tick Rhipicephalus annulatus, M. pingshaense and M. robertsii are intermediate in virulence, and M. majus is of low virulence. We transformed all four Metarhizium species to express GFP and used them in pathogenicity assays against diverse tick species. Key findings were that a) resistant ticks inhibit Metarhizium infection prior to hemocoel invasion by reducing fungal viability on the cuticle surface (Objective 2), as was supported by the in vitro studies of Objective 1, and b) Metarhizium kills susceptible ticks after cuticle penetration but prior to hemocoel colonization. Transcriptome studies of the most virulent species, M. brunneum, are in progress and include analyses of ungerminated conidia and conidia germination and development on a low nutrient medium or on susceptible R. annulatus exoskeleton (Objective 3). We anticipate these studies will contribute to identifying fungal genetic factors that increase virulence and speed of kill and may help reveal tick chemistries that could be included in biocontrol formulations to increase efficacy. Methodologies developed to screen tick cuticle extracts for ability to support conidia germination and development may help in the selection of wild fungi with increased virulence against resistant ticks. The overall knowledge gained should contribute not only to the improvement of tick control but also to the control of other blood-sucking arthropods and related plant pests. Use of bio-based agents for controlling arthropods will contribute to a healthier, more sustainable environment and serve a growing number of organic food farmers.