Presented as Thesis (Ph. D.)--University of Wisconsin--Madison, 1926. / Cover title. Reprinted from Phytopathology, vol. XVI, no. 10 (Oct. 1926). Includes bibliographical references (p. 695-696).

Thesis (M.Inst.Agrar.)(Microbiology)--University of Pretoria, 2005. / Title from opening screen (viewed March 20, 2006). Includes summary. Includes bibliographical references.

Competitive exclusion has been the mechanism hypothesized to account for the biological control of fire blight disease of pear and apple by the bacterium Pseudomonas fluorescens A506 (A506). Recent laboratory assays demonstrated, however, that A506 produces an antibiotic that is toxic to the fire blight pathogen, Erwinia amylovora, when cultured on media amended with iron (Fe����� or Fe�����). This study investigated this iron-dependent antibiosis by A506 by: 1) examining bioavailability of iron to A506 on blossom surfaces, 2) mutagenizing A506 to disrupt genes involved in antibiotic production, and 3) evaluating suppression of fire blight by A506 when co-treated with an iron chelate (FeEDDHA). Bioavailability of iron on blossoms was investigated with an iron biosensor [iron-regulated promoter (pvd) fused to an ice nucleation reporter gene (inaZ)] in A506. A506 (pvd-inaZ) expressed high ice nucleation activity (INA) on blossoms indicating a low-iron environment unlikely to induce antibiosis by A506. Spraying blossoms with FeEDDHA at concentrations ���0.1 mM significantly suppressed INA by A506 (pvd-inaZ). Transposon mutagenesis was used to generate and select mutants of A506 exhibiting altered antibiotic production profiles. One antibiotic-deficient mutant, A506 Ant���, was recovered; this mutant showed reduced epiphytic fitness on blossoms of apple and pear trees compared to the parent stain, A506. Another mutant, A506 Ant���, lost the characteristic fluorescent phenotype and exhibited iron-independent antibiotic production in defined culture media. A506 Ant��� established high populations on blossoms of apple and pear trees, similar to populations attained by A506, and reduced incidence of fire blight between 20 to 40%, levels comparable to A506 in orchard trials. In orchard trials, A506 was co-treated with FeEDDHA and fire blight suppression was evaluated. Bacterial strains established high populations on blossoms when co-treated with 0.1 mM FeEDDHA or in water. Significantly enhanced suppression of fire blight incidence by antibiotic producing strains of A506 amended with 0.1 mM FeEDDHA was observed in 2 of 5 trials, providing some evidence that iron-induced antibiosis can be a contributing mechanism in disease control. Lack of disease control by the antibiotic deficient strain, A506 GacS, and by 0.1 mM FeEDDHA alone added support to this hypothesis. / Graduation date: 2004

Graduation date: 1998

According to CDC, every year at least 2 million people are affected and 23,000 dies as a result of antibiotic resistance in U.S. It is considered one of the biggest threats to global health. More and more bacterial infections are becoming harder to treat. One such infection is fire blight, one of the most destructive disease of apple and pear trees. It is caused by bacteria Erwinia amylovora and its outbreaks have been known to destroy entire orchards in a single season. The conventional method of treatments includes use of antibiotics like streptomycin and oxytetracycline but the incidences like presence of multi-drug resistant bacteria in the mammals grazing in the fields have raised concerns. Phage therapy is considered one of the few ways available to combat bacterial resistance and prevent fire blight. In this method, a cocktail of highly lytic bacteriophages is prepared and sprayed on the trees at different time intervals. Bacteriophages are an “intelligent” drug. They multiply at the site of the infection until there are no more bacteria and then they are excreted back into the nature. These phenomena make them more efficient than an antibiotic, which kills all kind of bacteria including good bacteria and can be maintained in the environment for long periods of time. These qualities of bacteriophage have resulted in many commercially available phage therapies. The initial part of this research focuses on isolation, characterization and genomic comparison of bacteriophages that infect a plant pathogen E.amylovora of Erwiniaceae family of Enterobacteriales order. In this study, 28 novel bacteriophages were isolated, fully sequenced, characterized and grouped into seven families based on phage homology. To take this further, we characterized a novel jumbo family of bacteriophages that has a small burst size of 4.6-4.9 and are most similar to bacteriophages that infect Pseudomonas and Ralstonia rather than Enterobacteriales bacteria by protein similarity. These bacteriophages are shown to infect Erwinia and Pantoea bacterial strains, but no infection of 9 other bacterial strains tested, was seen, under laboratory conditions. The results of this work provide an insight on special characteristics that makes bacteriophage so unique and adaptable. The final part of this research explores the enormous diversity of bacteriophages. In 2014 Grose and Casjens grouped 337 fully sequenced tailed phages into 56 diverse clusters (32 lytic and 24 temperate). We further expanded our current understanding of these clusters by performing the comprehensive analysis of genomes and proteomes of 1037 tailed bacteriophages, posted on GenBank. The results of this work provide insights into diversity and relatedness of bacteriophages and the data is posted on GenBank.

Our present knowledge, as indicated by the above data, proves that fire blight is due to a specific organism, a bacterium known in science as Bacillus amylovorus, which manifests itself in the following ways: 1. Blossom blight, due to the bacterial infecting the nectary of the flower and multiplying therein, later passing down into the stem by way of the ovary and pedicel. Apparent by the browning of the flowers, which later become blackened. 2. Twig blight, due to inoculation infections through wounds made by insects or other agencies on the young and tender shoots, blighting from the tips downward, the leaves turning brown and appearing as though scorched by fire. 3. Fruit blight, due to the progress of the bacteria up the pedicel into the pulp of the fruit; appearing as brownish or much darkened areas, later involving the entire fruit. 4. Canker blight, due to the entering of the larger branches by bacteria, conveyed by insect, bird, or mechanical agents, or by the bacillus working back from infected twigs, fruit-spurs, or water-sprouts. These cankers vary in size from barely visible areas to a girdling of the entire limb; they appear roughened and depressed with a distinct line separating the canker from the apparently healthy tissue. They are called "hold-over cankers." 5. Collar blight is nothing more than canker blight which attacks the base of the trunk usually through an infected water-sprout, borers, or mechanical injury. Trees afflicted with collar blight soon take on the sickly appearance of half starved trees, prematurely defoliating (partially or wholly), and finally dying. 6. Leaf blight, due largely to insect injury, the majority of infections the margin, either lateral or terminal, although central lesions are found. The blighted portion of the leaf being a light or yellowish brown with a faint purplish border at the advancing edge, which when active shows a narrow watery zone. During the warm sunny days soon after rains, when the trees are in blossom and the pollen gathering insects are busy visiting the flowers, and the hold-over cankers are exuding drops of the blight bacterial, fire blight begins its havoc. Insects come in contact with this gummy substance and later visit the blossoms, thereby inoculating the flowers with the active organism, blossom blight resulting. Bees are awarded first place in spreading the blight to the flowers but flies and all other insects which visit the bloom are also liable to spread the germ. Later on in the season, as long as the tree is in a vigorous stage of growth, the aphids, ants, leaf hoppers, borers, beetles, and in fact any of the biting or sucking insects which inhabit the apple tree, may spread the organism to the succulent tissue, causing twig blight, fruit blight, leaf blight, or body blight. It is a fact that the insect, which in one section of the country is most troublesome in disseminating blight-bacillus, may not be so important an agent in another section. Different varieties of apple trees may also vary in different sections as to their powers of resistance or susceptibility; the York Imperial being listed as a susceptible variety in Pennsylvania and Virginia while in West Virginia it seems to be quite resistant. We cannot control weather conditions which play so important a part in the spread of blight. The weather may put a tree in a responsive or susceptible condition to inoculation and may also control insect life to a great extent. And it is indeed difficult to try to "harden up" a tree by the application of phosphate and potash fertilizers and by the discontinuing of cultivation, especially when the soil is strong and rains are frequent. Hence the more practical methods must be followed: 1. Fight the insects which spread the disease. Spray with nicotine sulphate or some other tobacco extracts in combination with the early scab or codling moth sprays. Where "Black leaf 40" is used, the recommended strength is 1 to 800 parts of water. 2. Cut out the source of infection,- the hold over cankers--cutting back an inch or two into healthy bark, and disinfect the wound with bichloride of mercury (1-1000). Prune out all blighted branches and if possible cut out all blighted twigs, cutting back several inches into the healthy wood. A severe winter often kills the germs in the twigs. All the waste material should be hauled out of the orchard and burned. 3. It is well to discontinue cultivation and application of manure or nitrogenous fertilizers as soon as blight its appearance. 4. The trees are most susceptible to the attack when planted in low wet ground; hence drain the low spots in the orchard. Many bulletins have stated that winter pruning is inducive to wood and thus favors fire blight. Little attention should be paid to such recommendations for we need trees with good growth which will carry a crop of fruit and allow the sun to penetrate throughout the tree and color up the apples. Many fruit growers have bearing orchards and are not anticipating new ones, so to such,the talk of susceptible and resistant varieties means little. Then too, some of the susceptible varieties are the best commercial apples to raise, and when such is the case it means careful inspection for the hold-over cankers. It is however well to pay some attention to this fact when planting an orchard. What is needed badly is community spirit or cooperation in this difficult work of controlling fire blight. For if one's neighbor does not keep the disease out of his orchard, it is sure to spread to the adjoining orchard. So everyone should "pull together" and control blight which has and is now causing the apple grower heavy losses. / Master of Science

Bacteria are ubiquitously present in every ecosystem on earth. While bacterial communities that reside in specific habitats, called the microbiota, have characteristic compositions, their constituents are exchanged between habitats. To understand the assembly processes and function of a microbial community in an ecosystem, it is thus important to identify its putative sources and sinks. The sources and sinks of the plant leaf microbiome, also called the phyllosphere microbiome, are still under debate. Here, I hypothesized that precipitation is a so far neglected source of the phyllosphere microbiome. Using 16S rRNA amplicon and metagenomic sequencing, I identified the genera Massilia, Sphingomonas, Methylobacterium, Pseudomonas, Acidiphilium, and Pantoea as members of the core rain microbiome in Blacksburg, VA. Further, I used rainwater as a bacterial inoculum to treat tomato plants. I showed that rain-borne bacteria of the genera Chryseobacterium, Enterobacter, Pantoea, Paenibacillus, Duganella, Streptomyces, Massilia, Shinella, Janthinobacterium, Erwinia, and Hyphomicrobium were significantly more abundant in the tomato phyllosphere 7 days post-inoculation, suggesting that these rain-borne bacteria successfully colonized the tomato phyllosphere and had a direct impact on the composition of its microbiome. These results were confirmed by comparing the phyllosphere microbiota of tomato plants grown under greenhouse conditions, and thus never exposed to rain, compared to plants grown outside under environmental conditions, including precipitation. Since a large diversity of bacteria is associated with rain, I also hypothesized that rain-borne bacteria are well adapted to environmental stresses, similar to the stressors microbial biopesticides are exposed to in the field. I thus explored rain as a source of resilient biopesticides to control fire blight, caused by the bacterial pathogen Erwinia amylovora, on apple. In an in-vitro dual culture assay, I identified rain-borne isolates displaying broad-range inhibition against E. amylovora and several other plant pathogens. Two rain-borne isolates, identified as Pantoea agglomerans and P. ananatis, showed the strongest inhibition of E. amylovora. Further experiments showed that these two Pantoea isolates survive under environmental conditions and have a strong protective effect against E. amylovora. However, protection from disease in an orchard was inconsistent, suggesting that the timing of application and formulations must be improved for field applications. Using a UV-mutagenesis screen and whole-genome sequencing, I found that a phenazine antibiotic produced by the P. agglomerans isolate was the likely active molecule that inhibited E. amylovora. Bacterial communities are constantly released as aerosols into the atmosphere from plant, soil, and aquatic sources. When in the atmosphere, bacteria may play crucial roles in geochemical processes, including the formation of precipitation. To understand the potential role of decaying vegetation as a source of atmospheric Ice Nucleation Particles (INPs), I analyzed a historic leaf litter sample collected in 1970 that had maintained Ice Nucleation Activity (INA) for 48 years. A culture-dependent analysis identified the bacterial species Pantoea ananatis and the fungal species Mortierella alpina to have INA and to be present in the leaf litter sample. Further, I determined that both P. ananatis and M. alpina produced heat-sensitive sub-micron INPs that may contribute to atmospheric INPs. The development of new sequencing technologies has facilitated our understanding of microbial community composition, assembly, and function. Most research in bacterial community composition is based on the sequencing of a single region of the 16S rRNA gene. Here, I tested the potential of culture-independent 16S rRNA sequencing of the phyllosphere microbiome for disease diagnosis. I compared the community composition of the microbiome of the aerial parts of cheddar pinks (Dianthus gratianopolitanus) that showed disease symptoms with the microbiome of healthy plants to identify the causative agent. However, I found that the pathogen is probably ubiquitous on cheddar pinks since it was present at similar abundance levels in symptomatic as well as healthy plants. Moreover, the low-resolution of 16S rRNA sequencing did not allow to identify the pathogen at the species or strain level. In summary, in this thesis, I found support for the hypothesis that rain is one of the sources of the phyllosphere microbiome, that rain is a promising source of biopesticides to control plant diseases in the field, that leaf litter is a source of atmospheric INPs, and that 16S rRNA sequencing is not well suited for pathogen identification in support of plant disease diagnosis. Finally, in additional research to which I contributed but that is not included in this thesis, I found that metagenomic sequencing can identify pathogens at the species and strain level and can overcome the limitations of 16S rRNA sequencing. / Doctor of Philosophy / Bacteria are present in nearly every ecosystem on earth. Bacterial communities that reside in a specific habitat are known as microbiota and have characteristic compositions and functions that directly impact the health of ecosystems. Microbiota associated with plants, the so-called plant microbiota, play a crucial role in plant fitness. Thus, it is important to study the assembly and diversity of plant microbiota and their impact on the ecosystem. The sources of leaf microbiota remain to be elucidated. Here, I have studied the contribution of rainfall to the bacteria that live on and in plant leaves. First, using DNA sequencing, I identified the bacteria present in rainfall in Blacksburg, VA. Then, using rain as bacterial inoculum, I found that some rain-borne bacteria, including members of the genera Pantoea, Massilia, Janthinobacterium, and Enterobacter, are efficient colonizers of tomato leaves. Either absence or low abundance of rain-borne bacteria from tomato leaves never exposed to rainfall confirmed further that bacteria in rain contribute to the assembly of plant leaf microbiota. The identification of all putative sources and sinks of leaf microbiota is important when trying to manipulate them to improve plant health and crop yield. Since I found that rainfall contains many different bacteria, I also studied the potential application of rain-borne bacteria in agriculture. The main limitations of commercial bio-pesticides are their poor survival and limited efficacy in the field. Here, I speculated that rain-borne bacteria are well adapted to environmental stressors and could represent efficient bio-pesticides under field conditions. In fact, I isolated two rain-borne bacteria from the genus Pantoea that strongly inhibited Erwinia amylovora, the causal agent of the fire blight disease of apple, in the laboratory under controlled conditions. However, I observed inconsistent results in a 2-year field trial in an orchard. Using mutagenesis and DNA sequencing, I found the active molecule that likely inhibited E. amylovora, in one of the rain-borne isolates. Finally, the access to newer and cheaper sequencing technologies has recently facilitated the study of bacteria at large scale. Most research of microbiota is based on the sequencing of a single region of one gene, the 16S rRNA gene. Here, I tested the potential of 16S rRNA sequencing of leaf microbiota for disease diagnosis. However, I identified the pathogen in healthy and diseased plants, suggesting its ubiquitous presence. Further, due to the low-resolution of 16S rRNA sequencing, it was impossible to identify the pathogen at the species level. In summary, I found that rain is a source that contributes to leaf microbiota, that rain is a promising source of bio-pesticides to control plant diseases, and that 16S rRNA sequencing is not recommended as a tool to diagnose plant diseases.

El fuego bacteriano, causado por Erwinia amylovora, es una enfermedad muy importante a nivel comercial y económico porque afecta a plantas de la familia de las rosáceas y es especialmente agresiva en manzano (Pyrus malus) y peral (Pyrus communis), así como en plantas ornamentales (Crataegus, Cotoneaster o Pyracantha). Esta enfermedad está distribuida por todo el mundo en zonas climáticas templadas de Amércia del Norte, Nueva Zelanda, Japón, Israel, Turquí y Europa. En España, el fuego bacteriano fue detectado por primera vez en 1995 en el norte del País (Euskadi) y más tarde en nuevos focos aparecidos en otras áreas. La enfermedad puede ser controlada comercialmente mediante la aplicación de pesticidas quimicos (derivados de cobre, antibioticos). Sin embargo, muchos de los productos químicos presentan baja actividad o causan fitotoxicidad, y la estreptomicina, el producto más eficaz, esta prohibido en muchos países, incluyendo España. Por tanto, en ausencia de apropiados agentes químicos, el control biológico se contempla como una buena alternativa. En el presente trabajo, un agente de control biológico, Pseudomonas fluorescens EPS62e, ha sido seleccionada de entre 600 aislados de las especies P. fluorescens y Pantoea agglomerans obtenidos de flores, frutos y hojas de plantas de la familia de las rosáceas durante una prospección llevada a cabo en varias áreas geográficas de España. La cepa ha sido seleccionada por su capacidad de suprimir la infecciones producidas por E. amylovora frutos inmaduros, flores y brotes de peral en condiciones de ambiente controlado, presentando unos niveles de control similares a los obtenidos mediante el control químico usando derivados de cobre o antibióticos. La cepa además ha mostrado la capacidad de colonizar y sobrevivir en flores y heridas producidas en frutos inmaduros en condiciones de ambiento controlado pero también en flores en condiciones de campo. La exclusión de E. amylovora medinate la colonización de la superficie, el consumo de nutrientes, y la interacción entre las células del patógeno y del agente de biocontrol es la principal causa de la inhibición del fuego bacteriano por la cepa EPS62e. Estas características constituyen aspectos interesantes para un desarrollo efectivo de la cepa EPS62e como un agente de biocontrol del fuego bacteriano en condiciones comerciales. / Fire blight, caused by Erwinia amylovora is a serious disease of rosaceous plants with great commercial and economic interest that is distributed over the world. Disease may be controlled commercially by the application of chemicals (copper compounds, antibiotics). Many chemical agents have low activity or cause phytotoxicity, and streptomycin, the most effective antibiotic, is not approved for use in many countries, including Spain. Therefore, in the absence of suitable chemical control agents, biological control could provide a useful alternative. In the present work, a biological control agent, Pseudomonas fluorescens EPS62e, has been selected among 600 isolates of P. fluorescens and Pantoea agglomerans obtained from flowers, fruits and leaves of rosaceous plants in a survey performed through several geographic areas of Spain. This strain has been selected for its capacity to suppress immature fruit, blossom and shoot infections caused by E. amylovora, under controlled environment conditions, providing control levels similar to chemical control with copper or antibiotic compounds. The strain has also shown the capacity to colonize and survive well in flowers and wounds on immature fruit under controlled environment conditions but also in flowers under natural conditions. Pre-emptive exclusion of the pathogen E. amylovora by surface colonization and nutrients depletion, and cell-to-cell interaction appear to be the main mechanisms of biocontrol. These characteristics constitute interesting traits for an effective development as a fire blight biological control agent under commercial conditions.

Pseudomonas fluorescens EPS62e es va seleccionar com a agent de biocontrol del foc bacterià per la seva eficàcia en el control de Erwinia amylovora. En aquest treball es van desenvolupar mètodes de traçabilitat que van permetre la seva detecció específica i quantificació. Mitjançant les tècniques RAPD i U-PCR es van obtenir fragments d'amplificació diferencial per EPS62e que es van seqüenciar i caracteritzar com marcadors SCAR per dissenyar una PCR en temps real. La PCR a temps real es va utilitzar simultàniament amb mètodes microbiològics per estudiar l'adaptabilitat epifítica de EPS62e en pomera i perera. L'ús combinat de mètodes microbiològics i moleculars va permetre la identificació de tres estats fisiològics de EPS62e: la colonització activa, l'entrada en un estat de viable però no cultivable, i la mort cel·lular. Aquest treball mostra que EPS62e està ben adaptada a la colonització de flors a camp, encoratjant la seva utilització dins d'una estratègia de control biològic contra el foc bacterià. / Pseudomonas fluorescens EPS62e was selected as a reliable biological control agent of fire blight for its high efficacy controlling Erwinia amylovora infections. In the present work, monitoring methods which allowed EPS62e specific detection and quantification were developed. RAPD and U-PCR fingerprints were used to obtain differential amplified fragments from EPS62e that were sequence characterized as SCAR markers. A real-time PCR was developed on the basis of the strain-specific SCAR markers, and was used simultaneously with microbiological methods to study the environmental fate of EPS62e in apple and pear orchards. The combined use of both microbiological and molecular methods permitted the identification of three physiological states for EPS62e, which consisted of active colonization, survival and entry into a viable but nonculturable state, and cell death. The present work shows that EPS62e is well adapted for blossom colonisation in the field, and encourages its utilisation in a fire blight.

Fire blight, caused by Erwinia amylovora, is a serious disease, which attacks plants within the family of Rosaceae. This bacterial disease causes major problems in the cultivation of pears (Pyrus communis) around the world. The occurrence of the disease is still geographically limited to certain areas in Sweden and neighbouring countries. However, increasing temperatures due to climate change and the relative unawareness of the public, augment the risk of its spread. Within the EU, there are no approved antibiotics for use in pear orchards. Within the species of P. communis, cultivars vary in their susceptibility to fire blight. Many scientists believe that new, resistant cultivars and rootstocks are important tools to enable pome fruit cultivation in the future.In this study,the susceptibility of some Swedish pear heirloom cultivars to E. amylovora was investigated by using immature pear fruit. The bacteria were introduced into immature pear fruit by using defined concentrations of inoculum. The fruit were incubated at 25°C, and disease severity was recorded over time. Disease was measured and recorded as the extent of lateral lesion size on the surfaces of the pear fruit. No definite differences in disease development were recorded across the concentrations tested. The cultivars were compared to one another, and ranked according to susceptibility. The most susceptible to the least susceptible were: `Sollerö´ > `Unknown Gävle´ = `Esperens herre´ = `Aspa´ > `Höstbergamott´ > `Göteborgs Diamant´ > `Conference´ = `Lilla dalpilen´ = `Carola´ > `Alexander Lukas´ = `Blodpäron´ = `Bonne Louise´. The results could have become clearer if immature fruit of younger physiological age had been used. Nevertheless, the ranking of some cultivars agrees with the published results of others.