Pseudomonas syringae: The Pathogen and Epiphyte

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Introduction

Fig 1. Electron Micrograph of Pseudomonas syringae. A rod shaped and gram-negative bacteria possessing polar flagella. http://tankerenemy.blogspot.com/2010/11/rain-making-bacteria-by-jay-hardy.html [2


Pseudomonas syringae is responsible for various functions within the microbial community and plays a diverse role in the biology of the phyllo-sphere as a pathogen, epiphyte, and ice nucleus.[1] The original strain was isolated in 1902, by van Hall, via diseased lilacs (Syringa vulgaris), directly corresponding to the species designation, syringae. By the early 1970s, nearly 40 stains of the phytopathogenic bacteria were isolated form varying plant species lesions and host specificity became an important criterion used by plant pathologist to differentiate variable species.[1] Overall,P. syringae is characterized as an aerobic, Gram-negative, rod-shaped bacterium with polar flagella (Fig. 1).[1] They do not accumulate poly-B-hydroxybutyrate, but do produce diffusible fluorescent pigments and associate as arginine dihydrolase and oxidase negative.[1] Additionally, with many variable strains, Phytobacteriologist have created a system for distinguishing between these bacteria via a species designation “pathovar” (pv.).[1] Also, as a plant-based pathogen known for its epiphytic abilities, P. syringae is also incredibly active in ice-nucleation.[3] With such a wide set of biological interactions and significant contributions to biological systems, understanding the molecular basis of P. syringae is critical and has resulted in P. syringae acting as a model for the study of host-pathogen interactions in various experimental hypotheses.[1] In addition, the abundance of P. syringae in rain, snow, and wild plants has been closely reported and corresponds with studies looking into P. syringae in relation to biochemical traits, pathogenicity and pathogenicity-related factors.[3] In all, P. syringae is an archetype of plant pathogens, an ubiquitous epiphyte with well-studied ecology, whose mechanisms of pathogenicity and evolution have been rigorously investigated.[3] With the majority of scientific investigations relating to this bacterium focused on its roles as a plant pathogen, the emergence of P. syringae and ice nucleation (IN) activity is also of critical importance.[3] With direct relation to processes in which the role of freezing is critical, investigations have led to the idea of P. syringae having an elaborate history whereby it survives and proliferates in diverse niches in habitats linked to the water cycle.[3] Therefore, the complex role of P. syringae within the microbial world can hold significant impacts on various environmental processes and biological systems.



Historical Context and Associated Taxonomy

Early History of the Genus

Fig 2. The P. syringae Complex. A visual list of all the Pseudosmonas species and pathovars considered within the P. syringae Complex. http://www.sipav.org/main/jpp/volumes/0410/041002.pdf [4


Pseudomonas was first understood to comprise all bacteria characterized as aerobic, Gram-negative rods with chemoorganotrophic metabolisms, and mobility from one or more flagella.[1] This wide association was only recently refined in the late 1800s through comparative analyses of 16S rDNA where the fluorescent, poly-B-hydroxybutyrate negative pseudomonads were grouped under Y-Proteobacteria and the non-fluorescent, poly-B-hydroxybutyrate positive pseudomonads were grouped in the B-Proteobacteria.[4] The Pseudomonas genus is one of the earliest in the Y-Proteobacteria class, where the evolutionally history is founded on hundreds of millions of years spend mostly in aquatic habitats under the absence of highly evolved plants and agriculture.[3] With a defined genus, the mid-1900s saw an increase in Pseudomonas species identification beginning with P. mori by Boyer and Lambert.[4] Up until the 1960’s it was thought that a major component of the physiology of pathogenic bacteria must be devoted to pathogenic activity where nutritional and cultural differences must reflect early evolutionary metabolic and genetic differences relating to pathogenicity.[4] Such early species proposals were cited on very minimal morphological, biochemical, and nutritional test along with colony appearance on differing media.[4] Thus, the assumption that specific ecological response involved large components of cell metabolism was generally assumed and thus resulted in an increase of named species as synonyms for the same pathogen.[4] This assumption was soon reconsidered with the origin of the P. syringae complex (Fig 2).[4]

The idea of a P. syringae complex developed to characterize as a single species comprising distinct populations capable of infecting limited ranges of hosts. [4] This idea began with a study of fifteen determinative tests that were considered to differentiate fluorescent plant pathogenic Pseudomonas.[4] Of the fifteen determinative tests, it was concluded that only five tests differentiated five distinct pathogenic species groups.[4] These tests include production of levan, capacity to rot potato, oxidase activity, production of arginine dihydrolase, and hypersensitivity reaction in tobacco (LOPAT). From these tests evolved LOPAT Group I pathogens, representing species that gave negative reactions in oxidase activity, capacity to rot potato, production of arginine dihydroalase, and positive hypersensitivity in tobacco.[4] However, Sands et al. (1970) established the idea that many of the LOPAT Group I species could not be explicitly identified or distinguished phenotypically or through biochemical and nutritional tests.[4] Additional DNA-DNA hybridization studies, performed by Palleroni et al. (1972) and Pecknold and Grogan (1973), determined genomic diversity with LOPAT Group I species but lacked significant results to adequately base taxonomic conclusions.[4] Therefore, with increased confusion among bacterial nomenclature, a general revision took place among the International Committee on the Systematics of Bacteria establishing the development of the Approved Lists of Bacterial Names and the International Standards for Naming Pathovars.[4]

As a consequence of these revision,P. syringae pathovars were established. Prior to the revisions, all LOPAT Group I species were grouped under a single species, P. syringae, as pathovars.[4]However, with further identification the P. syringae complex saw an expansion to include a broad quantity of closely related species (Fig 2).[4] This great diversity paired with phenotypic and genotypic variability is not singular to pathogenicity but is also cited in nutrient utilization patterns, bacteriophage sensitivity, bacteriocin production and sensitivity, ice nucleation activity, toxin production, copper and streptomycin resistance, plasmid profiles, and restriction fragment length polymorphism. [1]


Pathogenic Structure

Fig 3.DNA relatednessamong pathovars of P. syringae and related species. The values in the table represent DNA relatedness, expressed as percentage relative reassociation of the particular combination of 3H-labelled and unlabelled DNA. https://www.microbiologyresearch.org/docserver/fulltext/ijsem/49/2/ijs-49-2-469.pdf?expires=1617666697&id=id&accname=guest&checksum=D2F2F678A41DE881352F67532C87F9EB [5


After 1980, P. syringae encompassed a population composed of pathogenic strains, including a magnitude of sub-populations or pathovars, with highly restricted host ranges.[4] With a poorly understood structure of pathogenic populations within the P. syringae complex, the differentiation of distinguishable pathovars were previously all ascribed as host of P. syringae pv. syringae.[4] However, strains have been proven to be pathogenic to lilac, (the first isolated host) and also to over 44 other plant species, more so some pathovars with the same determinative characteristics were determined not to attack lilac.[4] This relates directly to the traditional, yet flawed, method of identification of pathogenic species by proving specific pathogenicity to a suspect host as previously described.[4]

It was not until after 1980 that new methods contemporaneously began development alongside the new nomenclature being applied to bacterial classification. New insights began with Wayne et al. (1987) who proposed a quantitative definition of a bacterial species as the population whose strains share more than 70% DNA-DNA hybridization when also paired with supporting phenotypic data.[4] An investigation by Gardan et al. (1999) identified nine “genomospecies” within the P. syringae complex via DNA-DNA re-association (Fig 3).[5] This genomospecies structure provides important insights into the various relationships within the complex. Additional polyphasic studies have been evaluated in order to differentiate Pseudomonas species via quinone systems, fatty acid, protein, polar lipids or even polyamine profiles.[4] However, results are only significant when species are undoubtedly distinct.

Following the classification struggles of the 1980s, the use of PCR primers offered a reliable method for the confirmation of pathovar identity. Palacio-Bielsa et al. (2009) established a list of thirty primes for the nineteen members of the P. syringae complex.[4] These primers include, the species P. avellanae, P. cannabina and P. fuscovaginae, and the pathovars actinidiae, alisalense, atropurpurea, coryli, glycinea, maculicola, morsprunorum, papulans, phaseolicola, pisi, savastanoi, sesami, syringae, tagetis, theae, and tomato.[4] However, comprehensive studies for a full list of primers is desirable and will aid in confirmation specificity.[4] Therefore, more recent focus has been on comparative sequencing analysis using appropriate genes paired with an evident relationship between selected genes that correspond to the overall genome.



Population Biology

Flexibility in Host Preferences

P. syringae populations exist within distinct microbial communities experiencing various weather conditions on the leaves of nearly every terrestrial plant species that grow across the globe. The population size of P. syringae can be defined as a function of four populational processes. These processes include growth, death, immigration, and emigration.[6] With the vast range of genotypes identified within the P. syringae species, it is reasonable to assume that growth and immigration equal or exceed death and emigration.[6] Thus, most strains of P. syringae can grow on multiple plant species, however one is often preferred and thus pathogenic strains of P. syringae will vary in regards to the plant species or family they infect.[6] These are identified as pathovars, which share many overlapping similarities among them. Also, for strains of P. syringae that are pathogenic or ice nucleation active, bacterial populations have the ability to affect disease or frost injury probability.[6] Therefore, a key focus and challenge is to determine the quantitative contributions of immigration, emigration, growth, and death to bacterial population sizes in order to associate biotic or abiotic factors that influence such processes.[6]


Fig 4. Qualitative variability in P. syringae bacterial populations on individual leaflets of field-grown snap bean (Phaseolus vulgaris) plants. Leaflets, sampled on the same day, were gently pressed onto King's medium B . https://www.ncbi.nlm.nih.gov/pmc/articles/PMC99007/ [6


Variability can be examined in terms of epiphytic host preference (Fig 4).[6] An epiphytic host can be characterized as a plant on which P. syringae can maintain a residency population in the absence of disease, it may or may not also serve as a host that P. syringae can infect.[6] In similarity to the idea that not all strains of P. syringae are pathogenic, some strains may only include epiphytic hosts.[6] This range of epiphytic hosts is commonly studied with concern with potential sources of inoculum for infection of susceptible hosts and are often isolated from weeds and other non-host plants in greenhouse or growth chamber studies.[6] Another key preference occurring among epiphytic populations of P. syringae relate to cultivar partiality.[6] Reports have shown association of lower epiphytic populations of pathovars of P. syringae on resistant compared to susceptible cultivars for several P. syringae-host systems.[6]

The measurement of epiphytic bacterial population sizes has been based on the ease with which culturable bacteria can be removed form leaves by either washing or sonication.[6] Epiphytic bacteria are often conceptualized as those that colonize the surface of leaves, what is actually important to measure are the culturable bacteria that can be removed.[6] Although it is probable that the majority of the bacteria in leaf washings or sonicates were on the surface (i.e. epiphytic) it is also important to assume the possibility that some bacteria in “protected sites’ on the epidermal layer were not so easily removed.[6] Thus, alternative means to measure the number of leaf-associated bacteria is to homogenize both the leaf and plate from the homogenate.[6] However, this method, while resulting in better bacterial recovery, provides no information relating to where on the leaves the bacteria originated.[6] Additionally, studies have shown that bacterial population sizes often follow a lognormal distribution across populations of leaves (Fig. 5).[6] Therefore, it can be stated that variability in bacterial population sizes are present not only among individual leaves but also at other sites on and in a single leaf.[6]

Fig 5. QuantitativevariabilityinpopulationsizesofP.syringaeonindividual leaves. (A) Each plate represents an equivalent dilution from washings of dif- ferent individual rye leaves. (B) Lognormal distribution of population sizes of P. syringae on two sets of individual bean leaflets. The mean population size for both sets of leaflets is approximately 5.0 log CFU/leaflet. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC99007/pdf/mr000624.pdf[6



Another variation in population size common of P. syringae include temporal variations, regardless of plant species, geographic area, or time scale.[6] On perennials, it has been proven that population sizes of P. syringae are generally higher in spring compared to summer months.[6] Additionally, on annual crops, population sizes may fluctuate form undetectable on young plants to greater than 10^7 colony forming units (CFU) per leaf on older plants.[6] Interesting, predictions of P. syringae population size may even be compared to forecasted weather patterns. Such short-term dynamics in population size can be studies using appropriate sampling frequencies. For example, generation times for several strains representing different P. syringae pathovars ranged from 1.13 to 2.25 hours in broth cultures at constant temperatures.[6] Additionally, Young et al. established that despite optimum temperature growth of four P. syringae strains was about 28 degrees Celsius, the doubling time of 1.27 hours a that temperature was insignificantly different from growth rates over the range of 23 to 33 degrees Celsius. Therefore, factors beyond temperature may limit development of varying P. syringae populations.[6] Additionally, a comparison of doubling times of P. syringae inside leaves with rates alongside healthy leaves may provide clues to gain understanding of the relative adaptation of this bacterium to an epiphytic as compare to a pathogenic existence.[6]

Furthermore, it has been shown that rates of population increase of P. syringae on snap bean leaves can lead to potentially severe brown spot disease hazards in just a few hours. Growth is considered the dominant population process in such snap bean leaves, and immigration (Fig. 6) is likely to be more important in delivering small amounts of P. syringae cells to aerial parts of plant.[6] Both death and emigration are thought to account for some of the rapid, large decrease that was observed in P. syringae population on snap bean leaves.[6] Significantly, a large decrease in P. syringae population sizes immediately following rain is believed to be attributed to emigration as a result of wash-off.[6]

Fig 6. Immigration and emigration of bacteria to snap bean canopies.(A) Emigration rates are based on measurements of concentrations of air-borne bacteria at canopy height and 1.5 m above the canopy using six-stage Andersen viable samplers. (B) Immigration was measured by the deposition of air-borne bacteria onto petri dishes filled to the rim with King's medium B. Measurements of immigration and emigration were made over canopies of snap beans during three growing seasons. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC99007/[6



As previously discussed, factors such as rainfall and wash-off can lead to varying effects on P. syringae population sizes. The larger population sizes accounted for on leaves and aerial parts of pome and stone-fruit trees during spring and autumn as compared to summer has been credited to cooler temperatures and frequent rainfall.[6] This positive association between rain and large population size has also been reported in annual herbaceous crops. Through evaluation of snap bean leaves, Hirano and Upper established that rainfall appears to be the greatest factor effecting temporal variability of P. syringae population sizes.[6] They determined population sizes of P. syringae decreased by 10- to 100-fold between days preceding and following rain, and thus a result of wash-off.[6] However, they also determined that after the initial decreases, population sizes of P. syringae increase suggesting rain can trigger the onset of rapid multiplication.[6] Interesting however, water in the form of fog, dew, mist or high humidity is not enough to provide sustainable growth conditions on snap bean leaves. [6] .

Epiphytic Bacterial Population Sizes and Disease

It is generally accepted that epiphytic population of P. syringae on asymptomatic leaves of susceptible or resistance host and non-host plants can function as a source of inoculum for disease.[6] One such example of a quantitative relationships seen between epiphytic populations sizes and subsequent disease is in P. syringae pv. syringae-snap bean-bacterial brown sport disease.[6] Studies, such as those by Lindemann et al., looking to quantify and evaluate this complex relationship looked at the amount of brown spot disease in relation to population size (determined via leaf washing) on dose-response in field experiments.[6] Results found that mean pathogen population sizes were not predictive of brown spot disease.[6] However, when the frequencies of epiphytic population sizes of P. syringae pv. syringae were equal or greater than 10^4 CFU per leaflet on asymptomatic induvial bean leaflets disease was able to be predicted.[6] Therefore, a quantitative relationship was established between epiphytic population sizes of P. syringae pv. syringae and the amount of disease that followed and thus the amount of disease may be the result of large bacterial population sizes when in association with leaves.[6]

With this critical information, it opens the question as to why P. syringae destroys its own habitat, through either lesions or frost injury.[6] Based on experimental reasoning, it is believed that lesions may provide a place for the bacterial community to survive during unfavorable weather conditions.[6] Also, it has been determined that population sizes are very variable within lesions, but have the ability to remain relatively large even after weeks of inadequate dry weather conditions.[6] This contrasts P. syringae associated with asymptomatic leaves which indicate a decline in relative population size under the same unfavorable conditions.[6] While no clear selection for habitat destruction has be determined, both lesion formation and frost injury have been proven to occur more when population sizes are large, suggesting previous bacterial success on or in their host.[6] Similarly, it has been shown that the true function of P. syringae is to live on healthy leaves, with possible means of communication with the host to improve habitat conditions without resulting damage.[6] Thus, it is only when conditions turn unfavorable and populations reach very large sizes that the system “crashes” to the detriment of both host and bacteria.[6]

P. syringae and Disease of Fruit Trees...A Closer Look


Fig. 7. Disease cycle of the bacterial canker pathogen Pseudomonas syringae pv. syringae. https://apsjournals.apsnet.org/doi/pdf/10.1094/PD-91-0004[7

Fruit-producing areas across the world are experiencing a major concern in regards to P. syringae diseases resulting in significant economic losses and exceeding difficultly in controllable measures. Systemic infection and death of young trees is a perennial problem associated with nurseries and canker development resulting in the death of scaffold limbs and entire trees.[7] Such infections have been seen with bacterial canker of plum caused by P. syringae pv. syringae in Germany, resulting in annual tree mortality rates as high as 30%, along with bacterial canker of hazelnut resulting in tree mortality across many European countries.[7] P. syringae is known to cause diseases of monocots, herbaceous dicots, and woody dicots. These pathogens use varying effectors, such as toxins, and phytohormones, to incite disease symptoms.[7]

Studies looking at taxonomic relationships determined P. syringae pathogenic to woody hosts are distributed among various groups and do not cluster together even when strains are isolated from the same species.[7] This suggests the lack of strains relations with the host is a common thread among all P. syringae; additionally, this observation was confirmed via a phylogenetic analysis using multilocus sequencing typing.[7] Thus, P. syringae pathogens of fruit trees are not necessarily distinct form P. syringae pathogens of herbaceous dicots and monocots.[7]

Fig. 8. Canker formation and enlarging cankers on sweet cherry caused by Pseudomo- nas syringae pv. syringae as a result of blossom blast and internal infection the previ- ous spring. https://apsjournals.apsnet.org/doi/pdf/10.1094/PD-91-0004[7


With varying Pseudomonas pathovars exhibiting similarities and differences among disease symptoms the life cycles of incited symptoms can be mapped.[7] Pseudomonas pathogens of fruit trees are seen to infect leaves, shoot tips, and fruit, along with unique infection and overwintering sites.[7] The three key fruit tree disease symptomatology caused by P. syringae include bacterial canker of stone fruit, olive knots, and apical necrosis of mango.[7] The development of bacterial cankers on stone fruit, whose formation will eventually girdle and kill plant branches resulting in fruit loss and tree death, can be facilitated by stress events such as exposure to freezing conditions and frost injury.[7] Additionally, P. syringae strains can enter woody tissue via wounds caused by pruning in highly commercial orchards, causing detrimental cankers formation.[7] The disease cycle of bacterial canker is initiated in the spring through the colonization and development of large P. syringae populations on blossoms (Fig. 7).[7] Olive knots result from hyperplasia formation on the stems and branches of the host plant, the accumulation of these knots can lead to severe tissue damage and the production of “off-tasting” fruit.[7] Similarly, bacterial apical necrosis of mango is characterized by rapid expansion of necrotic lesions on buds and leaves that can lead to severely debilitated trees or even tree death.[7]

Virulence factors are also important aspects of P. syringae disease association. Ice nucleation activity (INA) is evidently critical, yet a controversial topic, as freezing temperatures increase the severity of P. syringae pv. syringae infection of leaves in varying fruit tree species.[7] INA P. syringae growing epiphytically on frost sensitive leaves are associated with frost damage but not the existence of disease symptoms post-frost occurrence.[7] It is key to note that P. syringae pv. syringae is not linked directly to frost injury on blossoms but to subsequent invasion and tissue infection (Fig. 8).[7] Another key virulence factor includes effectors and the type III secretion system. The type III secretion system is an intimate interaction between pathogen and host plants where the pathogen is able to introduce effector proteins directly into the plant cells, functioning either to suppress host defense responses or to effect disease.[7] Furthermore, phytotoxins such as pore-forming toxins (syringomycin) and antimetabolites (tabtoxin) are used by Pseudomonas pathogens to target host plasma membrane and inhibit biosynthesis of amino acids.[7]

Overall, wood infection, overwintering sites within perennial host, and systemic movement through hosts via virulence factors distinguish P. syringae diseases of fruit trees and herbaceous plants. However, additional phylogenetic analysis suggests the typical clustering of fruit trees and herbaceous hosts of P. syringae pathogens indicates pathogenesis strategies that may intersect between pathogen groups.[7]


P. syringae and the Water Cycle

Conclusion

Fig 2. P. syringae.—pathogen, ice nucleus, and epiphyte. (A) Foliar and (B) pod symptoms of bacterial brown spot disease of snap bean (P. vulgaris) caused by P. syringae pv. syringae. (C) An ice nucleation event occurred in the test tube on the right due to the large numbers of ice nucleation-active P. syringae present on the leaf. (D) Symptoms of frost injury to snap bean plants in the field. (E).https://www.ncbi.nlm.nih.gov/pmc/articles/PMC99007/pdf/mr000624.pdf [9

References

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Authored for BIOL 238 Microbiology, taught by Joan Slonczewski, 2021, Kenyon College.