Epiphytic Bacteria and Yeasts on Apple Blossoms and Their Potential as Antagonists of Erwinia amylovora

Apple blossoms were sampled for indigenous epiphytic populations of culturable microorganisms during different stages of bloom at two locations in central Washington State and one site in Corvallis, OR. Frequencies and population sizes of bacteria on stigmas of apple were lower in Washington than at Corvallis, where average relative humidity was higher and possibly favored greater colonization; however, bacteria at Corvallis were mainly pseudomonads, whereas those in Washington were diverse, composed of several genera. In Washington, yeast as well as bacteria were isolated from both stigmatic and hypanthial surfaces. Sampled blossoms were processed immediately to assess microbial populations, or after a 24-h incubation at 28°C and high relative humidity, which broadened the range of detectable taxa evaluated as potential antagonists. Identifications were based on fatty acid methyl ester profiles and rDNA sequence analyses. Yeasts or yeastlike organisms were detected at frequencies similar to or greater than bacteria, particularly in hypanthia. When microbial isolates were tested for their capacity to suppress Erwinia amylovora on stigmas of detached crab apple flowers, many were ineffective. The best antagonists were the bacteria Pantoea agglomerans and Pseudomonas spp. and a few yeasts identified as Cryptococcus spp. Further evaluation of these taxa on flowers could lead to the discovery of additional biocontrol agents for fire blight.

Download Full-text

Control of Fire Blight by Pseudomonas fluorescens A506 and Pantoea vagans C9-1 Applied as Single Strains and Mixed Inocula

Phytopathology ◽

10.1094/phyto-03-10-0097 ◽

2010 ◽

Vol 100 (12) ◽

pp. 1330-1339 ◽

Cited By ~ 50

Author(s):

V. O. Stockwell ◽

K. B. Johnson ◽

D. Sugar ◽

J. E. Loper

Keyword(s):

Biological Control ◽

Pseudomonas Fluorescens ◽

Fire Blight ◽

Erwinia Amylovora ◽

Field Trials ◽

Disease Suppression ◽

Biological Control Agents ◽

Population Sizes ◽

Mixed Inocula ◽

Pantoea Vagans

The biological control agents Pseudomonas fluorescens A506 and Pantoea vagans C9-1 were evaluated individually and in combination for the suppression of fire blight of pear or apple in 10 field trials inoculated with the pathogen Erwinia amylovora. The formulation of pathogen inoculum applied to blossoms influenced establishment of the pathogen and the efficacy of biological control. Pantoea vagans C9-1 suppressed fire blight in all five trials in which the pathogen was applied as lyophilized cells but in none of the trials in which the pathogen was applied as freshly harvested cells. In contrast, Pseudomonas fluorescens A506 reduced disease significantly in only one trial. A mixture of the two strains also suppressed fire blight, but the magnitude of disease suppression over all field trials (averaging 32%) was less than that attained by C9-1 alone (42%). The two biological control agents did not antagonize one another on blossom surfaces, and application of the mixture of A506 and C9-1 to blossoms resulted in a greater proportion of flowers having detectable populations of at least one bacterial antagonist than the application of individual strains. Therefore, the mixture of A506 and C9-1 provided less disease control than expected based upon the epiphytic population sizes of the antagonists on blossom surfaces. We speculate that the biocontrol mixture was less effective than anticipated due to incompatibility between the mechanisms by which A506 and C9-1 suppress disease.

Download Full-text

Establishment of Bacterial Antagonists of Erwinia amylovora on Pear and Apple Blossoms as Influenced by Inoculum Preparation

Phytopathology ◽

10.1094/phyto.1998.88.6.506 ◽

1998 ◽

Vol 88 (6) ◽

pp. 506-513 ◽

Cited By ~ 52

Author(s):

V. O. Stockwell ◽

K. B. Johnson ◽

J. E. Loper

Keyword(s):

Fire Blight ◽

Erwinia Amylovora ◽

Culture Media ◽

Field Trials ◽

Bacterial Cells ◽

Fluorescent Microspheres ◽

Bacterial Antagonists ◽

Spray Inoculation ◽

Population Sizes ◽

Inoculum Preparation

The influence of inoculum preparation on the establishment of bacterial antagonists that suppress fire blight and Erwinia amylovora on blossoms was evaluated. Aqueous suspensions of Pseudomonas fluorescens A506, E. herbicola C9-1R, or E. amylovora 153N were prepared from cells harvested from the surface of an agar medium or from cells that were lyophilized after culture under similar conditions. Bacterial suspensions (1 × 108 CFU/ml) were sprayed on pear and apple trees at 50% bloom near midday. The incidence of recovery (proportion of blossoms containing detectable populations) and the population sizes of the bacteria on individual blossoms with detectable populations were followed over a period of several days. Fluorescent microspheres (1 μm in diameter) were added to sprays at a concentration of 1 × 107 microspheres per ml to mark blossoms that were open during application of bacteria. After dilution-plating, the stigmas and styles of each blossom were examined for the presence of microspheres with an epifluorescence microscope. In three of five trials, bacteria applied as suspensions of lyophilized cells were recovered from a greater proportion of blossoms than bacterial cells harvested directly from culture media. Every blossom harvested within 6 days after spraying had microspheres present on the surfaces of the styles and stigmas; thus, lack of establishment of detectable populations, rather than escape of blossoms from spray inoculation, accounted for the differences in proportion of blossoms colonized by the different preparations of bacteria. The use of lyophilized cells in field trials decreased variability in the establishment of bacteria on blossoms.

Download Full-text

Rates of Epiphytic Growth of Erwinia amylovora on Flowers Common in the Landscape

Plant Disease ◽

10.1094/pd-90-1331 ◽

2006 ◽

Vol 90 (10) ◽

pp. 1331-1336 ◽

Cited By ~ 6

Author(s):

K. B. Johnson ◽

T. L. Sawyer ◽

T. N. Temple

Keyword(s):

Growth Rates ◽

Fire Blight ◽

Erwinia Amylovora ◽

Prunus Avium ◽

Relative Growth ◽

Relative Growth Rates ◽

Scotch Broom ◽

Rubus Armeniacus ◽

Population Sizes ◽

Pollinating Insects

We evaluated epiphytic growth of the fire blight bacterium, Erwinia amylovora, on flowers of plant species common to landscapes where pears and apples are grown. The plants were from genera regarded as important nectar and pollen sources for pollinating insects: Acer, Amelanchier, Brassica, Cytisus, Populus, Prunus, Rubus, Salix, Taraxacum, Trifolium, and Symphoricarpos. Floral bouquets were inoculated with E. amylovora and incubated in growth chambers at 15°C for 96 h. Regardless of their susceptibility to fire blight, all species from the rose family except Prunus domestica (European plum) supported epiphytic populations of E. amylovora that exceeded 1 × 106 CFU/flower with relative growth rates for the populations that averaged 7% per hour. Nonrosaceous plants were generally poor supporters of epiphytic growth of the fire blight pathogen with relative growth rates averaging <4% per hour. In two seasons of field inoculations, the rosaceous non-disease-host plants, Prunus avium (sweet cherry) and Rubus armeniacus (Himalayan blackberry), yielded mean population sizes of E. amylovora that exceeded 1 × 106 CFU/flower; in contrast, at 8 days after inoculation, mean population sizes of the pathogen were in the range of 5 × 103 to 5 × 104 CFU/flower on Cytisus scoparius (Scotch broom) and <1 × 102 CFU on Acer macrophylum (big leaf maple). Because vectors of E. amylovora, principally bees, visit many kinds of flowers in landscape areas between pear and apple orchards, flowers of rosaceous, non-disease-host species could serve as potential sites of inoculum increase during their periods of bloom.

Download Full-text

Effect of rootstock clones on fire blight susceptibility in scion apple cultivars

Plant Protection Science ◽

10.17221/10552-pps ◽

2017 ◽

Vol 38 (SI 2 - 6th Conf EFPP 2002) ◽

pp. 552-554

Author(s):

J. Korba ◽

K. Patáková ◽

V. Kůdela

Keyword(s):

Relative Humidity ◽

Fire Blight ◽

Erwinia Amylovora ◽

Shoot Length ◽

High Relative Humidity ◽

Golden Delicious ◽

Apple Cultivars

This study was performed on 24 clonal rootstocks and 4 scion apple cultivars to evaluate rootstock/cultivar combinations for susceptibility to fire blight (Erwinia amylovora). For this aim, the clonal rootstocks were planted in plastic greenhouse in 1992 and grafted with 4 cultivars (Golden Delicious, Dulcit, Domino and Dione) in 1998. A total of 96 rootstock/cultivar combinations were tested during 1998–2001. Each year, 10 to 30 actively growing shoots per one combination were inoculated with the pathogen. The shoots were inoculated by cutting through first unfolding leaves with scissors dipped into inoculum (109 cfu/ml). The trees were misted to create a high relative humidity. The susceptibility of the test roostock/cultivar combinations was evaluated by calculating the length of necrosis to the total shoot length 40 days after inoculation. The degree of susceptibility of cultivar tested varied depending on the rootstocks and years. In order of decreasing to increasing levels of fire blight susceptibility of four cultivars tested, the pattern observed was Dulcit, Golden Delicious, Domino (moderately susceptible) and Dione (very susceptible). The lowest susceptibility of four cultivars tested was observed on TE 52 rootstock (on average, the cultivars were evaluated as intermediate), the highest susceptibility was on JTE-E and M9 rootstocks (the cultivars were very susceptible). The influence of 21 remaining rootstocks on susceptibility in scion cultivars was not marked. Thus some rootstocks can render the scion cultivars more fire blight susceptible or more resistant.

Download Full-text

Implications of Pathogenesis by Erwinia amylovora on Rosaceous Stigmas to Biological Control of Fire Blight

Phytopathology ◽

10.1094/phyto-99-2-0128 ◽

2009 ◽

Vol 99 (2) ◽

pp. 128-138 ◽

Cited By ~ 22

Author(s):

K. B. Johnson ◽

T. L. Sawyer ◽

V. O. Stockwell ◽

T. N. Temple

Keyword(s):

Biological Control ◽

Population Size ◽

Fire Blight ◽

Erwinia Amylovora ◽

Fluorescent Protein ◽

Virulent Strain ◽

Avirulent Strain ◽

Bacterial Antagonists ◽

Population Sizes ◽

Green Fluorescent

As a prerequisite to infection of flowers, Erwinia amylovora grows epiphytically on stigmas, which provide a conducive habitat for bacterial growth. Stigmas also support growth of several other bacterial genera, which allows for biological control of fire blight; although, in practice, it is very difficult to exclude E. amylovora completely from this habitat. We investigated the dynamics of growth suppression of E. amylovora by comparing the ability of virulent and avirulent strains of E. amylovora to compete with each other on stigmas of pear, apple, and blackberry, and to compete with a co-inoculated mixture of effective bacterial antagonists. When strains were inoculated individually, virulent E. amylovora strain Ea153N attained the highest population size on stigmas, with population sizes that were approximately double those of an avirulent hrpL mutant of Ea153 or the bacterial antagonists. In competition experiments, growth of the avirulent derivative was suppressed by the antagonist mixture to a greater extent than the virulent strain. Unexpectedly, the virulent strain enhanced the population size of the antagonist mixture. Similarly, a small dose of virulent Ea153N added to inoculum of an avirulent hrpL mutant of Ea153 significantly increased the population size of the avirulent strain. A pathogenesis-gene reporter strain, Ea153 dspE::gfp, was applied to flowers and a subset of the population expressed the green fluorescent protein while growing epiphytically on stigmas of apple. These results are consistent with the hypothesis that virulent E. amylovora modifies the epiphytic habitat presented by the stigma through a pathogenesis-related process, which increases host resources available to itself and, coincidentally, to nonpathogenic competitors. Over nine orchard trials, avirulent Ea153 hrpL significantly suppressed the incidence of fire blight four times compared with six for the antagonist mixture. The degree of biological control achievable with an avirulent strain of E. amylovora likely is limited by its inability to utilize the stigmatic habitat to the same degree as a virulent strain.

Download Full-text

In-orchard population dynamics of Erwinia amylovora on apple flower stigmas

Phytopathology ◽

10.1094/phyto-01-21-0018-r ◽

2022 ◽

Author(s):

Suzanne Slack ◽

Jeff Schachterle ◽

Emma Sweeney ◽

Roshni Kharadi ◽

Jingyu Peng ◽

...

Keyword(s):

Population Dynamics ◽

Wind Speed ◽

Relative Humidity ◽

Fire Blight ◽

Erwinia Amylovora ◽

Field Studies ◽

Post Inoculation ◽

Time Period ◽

Weather Parameters ◽

Frequent Sampling

Populations of the fire blight pathogen Erwinia amylovora Ea110 on apple flower stigmas were tracked over the course of apple bloom in field studies conducted between 2016 and 2019. In 18 of 23 experiments, flower stigmas inoculated on the 1st day of opening were found to harbor large (106-107 cells / flower) populations of E. amylovora when assessed three to five days post-inoculation. However, populations inoculated on stigmas of flowers that were already open for three days did not reach 106 cells / flower, and populations inoculated on stigmas of flowers that were already open for five days never exceeded 104 cells / flower. During this study, >10-fold increases in E. amylovora stigma populations in a 24-hr time period (termed population surges) were observed on 34.8%, 20.0%, and 4.0% of possible days on 1-day, 3-day, and 5-day open flowers, respectively. Population surges occurred on days with average temperatures as high as 24.5°C and as low as 6.1°C. Experiments incorporating more frequent sampling during days and overnight revealed that many population surges occurred between 10:00 PM and 2:00 AM. A Pearson’s correlation analysis of weather parameters occurring during surge events indicated that population surges were significantly associated with situations where overnight temperatures either increased or remained constant, where wind speed decreased, and where relative humidity increased. This study refines our knowledge of E. amylovora population dynamics and further indicates that E. amylovora is able to infect flowers during exposure to colder field temperatures than previously reported.

Download Full-text

Biological Control Agents for Fire Blight of Apple Compared Under Conditions Limiting Natural Dispersal

Plant Disease ◽

10.1094/pdis.2002.86.6.639 ◽

2002 ◽

Vol 86 (6) ◽

pp. 639-644 ◽

Cited By ~ 49

Author(s):

P. L. Pusey

Keyword(s):

Fire Blight ◽

Erwinia Amylovora ◽

Disease Incidence ◽

Field Trials ◽

Microbial Populations ◽

Biological Control Agents ◽

Two Factors ◽

Statistical Separation ◽

Natural Dispersal ◽

Potential Use

The efficacy of Pantoea agglomerans strain E325 for control of fire blight of apple was determined in comparative field trials involving other bacterial antagonists. Concurrently, the importance of the natural dispersal of bacteria as a complicating factor was assessed. Tests were performed under two sets of conditions, those that allowed for the dispersal of bacteria via honeybee activity and those that minimized it through the use of translucent polyethylene enclosures around single trees. The enclosures also raised daytime temperatures and allowed for controlled wetting, two factors important to the development of blossom blight. Singleantagonist treatments with strain E325, Pseudomonas fluorescens strain A506, and P. agglomerans strain C9-1 were applied to open blossoms on each of 10 enclosed trees and 10 nonenclosed trees. During bloom, suspensions of antagonists (108 CFU/ml) were applied twice with a brush, and a suspension of Erwinia amylovora (107 CFU/ml) was subsequently applied once using the same method. Two days after inoculation with the pathogen, trees were misted to simulate precipitation. Flower-to-flower spread of antagonistic bacteria was less frequent on trees surrounded by plastic enclosures than on nonenclosed trees. The range and statistical separation of means for population size of E. amylovora and disease incidence among treatments were greater for enclosed trees than for nonenclosed trees. Based on these results, the natural spread of antagonists being compared may mask differences in their efficacy as biocontrol agents. Such distortions and resulting misinterpretations could be lessened by separating treatments widely in large orchard blocks and by monitoring microbial populations. Strain E325 from fresh or lyophilized cultures was consistently more effective than standard antagonists in suppressing E. amylovora and reducing disease incidence. To fully assess its potential use for fire blight, larger-scale trials under various conditions will be necessary.

Download Full-text