Bacterial volatile organic compounds (VOCs) are signal molecules that may have beneficial roles in the soil-plant-microbiome ecosystem. In this Ph.D. thesis, I aimed to assess and characterize the role of bacterial VOCs in plant tolerance to drought and in the biocontrol of fungal pathogens. I started by studying two root endophytic bacteria isolated from pepper plants cultivated under desert farming conditions. They showed an enhancement of pepper tolerance to drought stress and an amelioration of its physiological status. Moreover, they induced the expression of a vacuolar pyrophosphatase proton pump (V-PPase), implicated in the regulation of the vacuolar osmotic pressure, facilitating water uptake. Besides, the exposure of Arabidopsis thaliana plants, grown under salinity stress, to the volatile 2,3-butanediol, described for its plant growth promotion (PGP) potential, enhanced the plants tolerance to salinity, proving the potential involvement of this volatile in the osmotic stress resistance mechanism. Then, I studied VOCs released by three bacteria associated to healthy rice plants. Their released VOCs mixtures modified the color pattern of Magnaporthe oryzae, the agent of the rice blast disease, and protected rice from the pathogen infection. A significant reduction of melanin production, sporulation and appressoria formation was measured in presence of the bacterial VOCs, without major effects on mycelial proliferation. 1-butanol-3-methyl, one of the nine VOCs co-produced by the studied bacteria, proved its potential of reducing M. oryzae melanin in vitro. In vivo tests confirmed the infection inhibition effects mediated by the rice-bacterial VOCs, with a reduction of 94% of the disease incidence. Lastly, I compared the genomes of the five bacteria considered in the previous experimental studies. The PGP traits and the VOCs pathways identified from the genome analyses confirmed the effects observed with the in vitro and in vivo assays, revealing a complex mode of promotion and protection offered by the studied plant-associated bacteria. In conclusion, plant-associated bacterial VOCs can play potentially important roles in modulating plant drought tolerance and reducing fungal virulence. Such biological resources represent novel tools to counteract the deleterious effects of abiotic and biotic stresses and have the potential to be exploited for sustainable approaches in agriculture.

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Fungal diseases, Pythium ultimum (Pythium leak) and Helminthosporium solani (silver scurf) have detrimental effects on potato tuber (Solanum tuberosum) quality and yield. Tubers are the world’s fourth largest agricultural food crop and are crucial for feeding a growing population. Bacteria from the genus Streptomyces are known for producing a wide variety of secondary metabolites with antifungal properties. Isolates of Streptomyces have recently shown inhibitory effects towards P. ultimum and H. solani in Petri dish assays. These data suggest that Streptomyces may work as a biocontrol to protect tubers from P. ultimum and H. solani. We tested talc-based powder formulas for their ability to maintain viable Streptomyces spores in storage. The formula that maintained spores the longest was then used to coat varying Streptomyces isolates onto a tuber surface that contained, or would be exposed to P. ultimum or H. solani. Tests were conducted in a lab, greenhouse, and field setting. We found a powder formula that kept 50% of the added Streptomyces spores viable for a period of three to six months depending on the isolate. Isolates with inhibitory effects towards H. solani were applied as a powder on seed tubers infected with H. solani and grown in a greenhouse. Upon completion of the experiment, we found that progeny tubers from neither the treatment nor the control groups contained H. solani. Instead, we found a similar surface pathogen, Colletotrichum coccodes (Black dot), on many of the progeny tubers. While not the target pathogen of this study, some isolates significantly limited C. coccodes compared to the control. This experiment was repeated in a field setting where C. coccodes was again the primary disease found on the progeny tubers. In the field, isolates showed no inhibitory effect towards C. coccodes. Isolates with inhibitory effects towards P. ultimum were applied as a powder onto wounded tubers. One hour later the tubers were exposed to P. ultimum. Isolates did not limit P. ultimum compared to the control after a week of incubation. A follow up experiment revealed that the Streptomyces isolate used needed at least 24 hours of growth to produce antifungal secondary metabolites. Our data suggest that Streptomyces bacteria can easily be stored in a powder and that there are beneficial effects as a biocontrol against C. coccodes. Our data also suggest that timing Streptomyces application for maximum secondary metabolite production may improve its efficacy as a biocontrol.

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Burkholderia contaminans MS14 shows excellent antimicrobial activities against a wide range of pathogens. Complete sequence analysis reveals that the MS14 genome harbors multiple gene loci that contribute to its antimicrobial activities and lacks key virulence features commonly found in pathogenic Burkholderia species. A mutagenesis study identified the genes required for MS14 antibacterial activities and gene expression profiling targeted a polyketide synthase (PKS) gene cluster. Site-specific mutagenesis confirmed the PKS gene cluster is directly related to MS14 antibacterial activities and the PKS gene product is predicted to be the MS14 antibacterial compound. Strain UFB2 isolated from Mississippi shows significant antifungal and antibacterial activities. UFB2 was classified to be Pseudomonas chlororaphis and its complete genome sequence was reported in this study. Green house trails showed P. chlororaphis strain UFB2 could efficiently reduce the disease severity of bacterial canker of tomato, by significantly inhibiting the growth of the pathogen Clavibacter michiganensis subsp. michiganensis. The research findings of B. contaminans MS14 and P. chlororaphis UFB2 have provided insights into the development of MS14 antibacterial compound for agricultural application and potential use of strain UFB2 as a biocontrol agent.

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Teniendo en cuenta el número de industrias alimentarias afectadas por el desarrollo de biofilms, es imprescindible un análisis integral e interdisciplinario de los factores que intervienen en su formación a fin desarrollar estrategias de remoción o prevención de estos. En esta tesis se propuso como objetivo principal profundizar el conocimiento sobre la adhesión, formación y producción de metabolitos en los biofilms formados por levaduras, principales deteriorantes de la industria productora de jugos, en presencia de bacterias patógenas pertinentes a la seguridad de los jugos de frutas, a fin de evaluar posibles estrategias de biocontrol. En el Capítulo I, se analizaron los biofilms multiespecie formados por levaduras y su asociación con bacterias patógenas, basándose en la hipótesis de que las levaduras presentes en la microbiota residente de las superficies de los equipos de producción de jugos de frutas pueden favorecer la persistencia de las bacterias a través de la formación de biofilms. Inicialmente, se llevaron a cabo estudios de los parámetros relacionados con la formación de biofilms de Listeria monocytogenes. Los resultados demostraron que Listeria exhibe un carácter altamente hidrofóbico, es móvil, produce curli y celulosa, es viable en jugo de manzana y se adhiere al acero inoxidable (AI) a 25 oC. Seguidamente, se describieron los biofilms formados por Rhodotorula mucilaginosa, Candida tropicalis, Candida krusei y Candida kefyr, en presencia de L. monocytogenes, Salmonella enterica o Escherichia coli O157:H7, sobre AI y membranas de ultrafiltración (MUF), empleadas para la clarificación de los jugos. Los análisis sobre los biofilms se realizaron empleando técnicas de recuento en placa, microscopía de fluorescencia y microscopía electrónica de barrido. Los resultados revelaron que las estructuras levaduriformes (pseudohifas, blastoporas) proporcionan un entorno propicio para el anclaje y la adhesión de las bacterias patógenas, convirtiéndose así en un nicho elegible para su permanencia en un ambiente que simula la producción de jugos de frutas. En el Capítulo II, se exploró la actividad inhibitoria y antibiofilm de Lacticaseibacillus rhamnosus GG ATCC 53103 (LGG) y Lactobacillus casei ATCC 393 frente a L. monocytogenes, S. enterica y E. coli O157:H7. Se evaluaron dos estrategias de biocontrol: competencia, en la cual tanto las células de Lactobacillus como las bacterias patógenas colonizan simultáneamente la superficie de AI; y exclusión, donde se forma un biofilm protector con células de Lactobacillus sobre la superficie de AI previo a la incorporación de las bacterias patógenas. Ambas especies de Lactobacillus: presentaron alta capacidad adhesiva sobre AI, sin alterar los parámetros fisicoquímicos del jugo de manzana. Además, estas cepas mitigaron la proliferación y adhesión de las bacterias patógenas hasta niveles no detectables en AI. Estos resultados sugieren que las cepas de Lactobacillus estudiadas podrían ser candidatas prometedoras para la formación de biofilms protectores en entornos de producción de jugos, ofreciendo una estrategia efectiva para controlar la adhesión microbiana en superficies relacionadas con la industria alimentaria. Con el objetivo de reducir o prevenir la colonización de las levaduras en las superficies y, de este modo, evitar la adhesión de bacterias patógenas que puedan entrar al sistema de producción de jugos de frutas, en el Capítulo III se evaluó la eficacia de moléculas quorum sensing (MQS) producidas por levaduras y del agente antifúngico natamicina (NAT), que se utiliza habitualmente en la industria alimentaria. En primer lugar, se identificaron compuestos orgánicos volátiles (VOC) en biofilms de levaduras, y cuantificaron en presencia y ausencia de bacterias patógenas afectaba su producción. La técnica de extracción con disolventes y la micro extracción en fase sólida se emplearon para la extracción de COV, seguida de un análisis mediante cromatografía gaseosa acoplada a espectrometría de masas. Los resultados demostraron que los biofilms de C. tropicalis producen farnesol (FAR), tirosol (TIR) y 2- feniletanol (2-FE), tres compuestos reconocidos como MQS. Por otro lado, la presencia de las bacterias patógenas no afectó significativamente (p>0,05) la producción de los COV liberados por las levaduras. En segundo lugar, se analizó el efecto de farnesol 0,06 mM (FAR) en combinación con natamicina 0,01 mM (NAT) sobre biofilms de levaduras y multiespecie formados sobre AI y MUF. Los biofilms tratados con NAT+FAR mostraron inhibición de la filamentación de las levaduras y una alteración significativa de la estructura tridimensional tanto en AI como en MUF. Además, se observó una disminución en la adhesión de L. monocytogenes, S. enterica y E. coli O157:H7 en los biofilms multiespecie. La combinación NAT+FAR podría considerarse un agente de control prometedor para prevenir la formación de biofilms en las líneas de procesamiento de jugo de manzana. / Considering the number of food industries affected by the development of biofilms, a comprehensive and interdisciplinary analysis of the factors involved in their formation is essential to develop strategies for their removal or prevention. The main objective of this thesis was to deepen the knowledge of the adhesion, formation, and production of metabolites in biofilms formed by yeasts, the main spoilage agents in the juice production industry, in the presence of pathogenic bacteria relevant to the safety of fruit juices, to evaluate possible biocontrol strategies. Chapter I examined the association between yeasts and foodborne pathogens in biofilms multispecies, based on the hypothesis that yeasts present in the resident microbiota on the surfaces of fruit juice production equipment may favour the persistence of bacteria through the formation of biofilms. Initially, studies were carried out on parameters related to biofilm formation of Listeria monocytogenes. Results indicate that Listeria has a highly hydrophobic character, is mobile, produces both curli and cellulose, is viable in apple juice, and adheres to stainless steel (SS) at 25 °C. Biofilms formed by Rhodotorula mucilaginosa, Candida tropicalis, Candida krusei, and Candida kefyr in the presence of L. monocytogenes, Salmonella enterica or Escherichia coli O157:H7 on SS and ultrafiltration membranes (UFM), were described. Biofilm analyses were performed using plate counting, fluorescence microscopy and scanning electron microscopy techniques. The results revealed that yeast-like structures (pseudohyphae, blastopores) supply a favourable environment for adhesion and adhesion of pathogenic bacteria, thus becoming an eligible niche for their permanence in an environment that simulates fruit juice production. Chapter II aimed to investigate the inhibitory and antibiofilm efficacy of Lacticaseibacillus rhamnosus GG ATCC 53103 (LGG) and Lactobacillus casei ATCC 393 against L. monocytogenes, S. enterica and E. coli O157:H7. Two biocontrol strategies were evaluated: competition, in which Lactobacillus cells and pathogenic bacteria colonise the SS surface at the same time; exclusion, in which a protective biofilm with Lactobacillus cells is formed on the SS surface and pathogenic bacteria are after incorporated. Both Lactobacillus species adhered to AI, without altering the physicochemical parameters of the apple juice. Furthermore, these strains mitigated the proliferation and adhesion of pathogenic bacteria to undetectable levels on AI. These results suggest that the studied Lactobacillus strains could be promising candidates for the formation of protective biofilms in juice production environments, offering an effective strategy to control microbial adhesion on food industry-related surfaces. To reduce or prevent yeast colonization on surfaces and thus avoid the adhesion of pathogenic bacteria that may enter the fruit juice production system, the efficacy of quorum sensing molecules (QSM) produced by yeasts and the antifungal agent natamycin (NAT), which is commonly used in the food industry, was evaluated in Chapter III. First, it was identified and quantified the volatiles organic compounds (VOC) released by the yeast biofilms, and then, it was assessed whether the presence of pathogenic bacteria affected the production of these metabolites. The solvent extraction technique and solid phase microextraction were used for metabolite extraction, followed by analysis by gas chromatography coupled to mass spectrometry. The results showed that C. tropicalis biofilms produce farnesol (FAR), tyrosol (TIR) and 2-phenylethanol (2-FE), recognized as MQS. On the other hand, the presence of the pathogenic bacteria did not significantly (p>0.05) alter the production of the VOC released by the yeasts. Second, it was evaluated the effect of farnesol 0.06 mM with NAT 0.01 mM on yeast and multispecies biofilms formed on SS and UFM. Biofilms treated with NAT+FAR showed inhibition of yeast filamentation and a significant alteration of the three-dimensional structure of both AI and MUF. In addition, decreased adhesion of L. monocytogenes, S. enterica and E. coli O157:H7 was observed in the multispecies biofilms. The NAT+FAR combination could be considered as a promising control agent to prevent biofilm formation in apple juice processing lines.

Mole Crickets (Scapteriscus spp.) are the most destructive pests in southern turf and pasture grasses. In response to extensive losses from mole crickets, Florida formed a task force to identify natural enemies in the native range of these pests. Two parasitic insects, Larra bicolor and Ormia depleta, and Steinernema scapterisci, an entomopathogenic nematode, were imported and released. Of the two insects, only Larra bicolor has spread to other states infested with mole crickets in the southeast. The present study documents the seasonal biology of Larra bicolor in the northern Gulf region, ornamental plants that can be used as nectar sources, and the impact of these nectar sources on longevity of the wasp and parasitism of mole crickets. Results of field and laboratory experiments showed that the ornamental Pentas lanceolata attracted wasps in the field and provided comparable or better longevity than Spermacoce verticillata which was the only known nectar source.