Development of a bacteriophage-based biopesticide for fire blight

Fire blight is an economically important disease of apples and pears that is caused by the bacterium Erwinia amylovora. Control of the disease depends on limiting primaly blosson1 infection in the spring, and rapidly removing infected tissue. The possibility of using phages to control E.amylovora populations has been suggested, but previous studies have. failed to show high treatment efficacies. This work describes the development of a phage-based biopesticide that controls E. amylovora populations under field conditions, and significantly reduces the incidence of fire blight. This work reports the first use ofPantoea agglomerans, a non-pathogenic relative ofE. amylovora, as a carrier for E. amylovora.phages. Its role is to support a replicating population of these phages on blossom surfaces during the period when the flowers are most susceptible to infection. Seven phages and one carrier isolate were selected for field trials from existing collections of 56 E. amylovora phages and 249 epiphytic orchard bacteria. Selection of the . /' phages and carrier was based on characteristics relevant to the production and field perfonnance of a biopesticide: host range, genetic diversity, growth under the conditions of large-scale production, and the ability to prevent E. amylovora from infecting pear blossoms. In planta assays showed that both the phages and the carrier make significant contributions to reducirig the development of fire blight symptoms in pear blossoms. Field-scale phage production and purification methods were developed based on the growth characteristics of the phages and bacteria in liquid culture, and on the survival of phages in various liquid media. Six of twelve phage-carrier biopesticide treatments caused statistically signiflcant reductions in disease incidence during orchard trials. Multiplex real-time PCR was used to simultaneously monitor the phage, carrier, and pathogen populations over the course of selected treatments. In all cases. the observed population dynamics of the biocontrol agents and the pathogen were consistent with the success or failure of each treatment to control disease incidence. In treatments exhibiting a significantly reduced incidel1ce of fire blight, the average blossom population ofE.amylovora had been reduced to pre-experiment epiphytic levels. In successful treatments the phages grew on the P. agglomerans carrier for 2 to 3 d after treatment application. The phages then grew preferentially on the pathogen, once it was introduced into this blossom ecosystem. The efficacy of the successful phage-based treatnlents was statistically similar to that of streptomycin, which is the most effective bactericide currently available for fire blight prevention. The in planta behaviour ofE. amylovora was compared to that ofErwinia pyrifoliae, a closely related species that causes fire blight-like synlptoms on pears in southeast Asia. Duplex real-time PCR was used to monitor the population dynamics of both species on single blossonls. E. amylovora exhibited a greater competitive fitness on Bartlett pear blossoms than E. pyrifoliae. The genome ofErwinia phage Ea21-4 was sequenced and annotated. Most of the 8-4.7 kB genome is substantially different from previously described sequences, though some regions are notably similar to Salmonella phage Felix 01 . Putative functions were assigned to approximately 30% of the predicted open reading frames based on amino acid sequence comparisons and N-terminal sequencing of structural proteins.

Tintype of Unidentified African American Woman Leaning on Chair [n.d.]

A small tintype of a young Black woman dressed in white. The date, location and name of the photographer are unknown. This tintype was among the family memorabilia belonging to Iris Sloman Bell, of St. Catharines, Ontario. Relatives of the Sloman - Bell family are descended from former American slaves."Tintypes were the invention of Prof. Hamilton Smith of Ohio. They begin as thin sheets of iron, covered with a layer of black paint. This serves as the base for the same iodized collodion coating and silver nitrate bath used in the ambrotype process. First made in 1856, millions were produced well into the twentieth century. When tintypes were finished in the same sorts of mats and cases used for ambrotypes, it can be almost impossible to distinguish which process was used without removing the image to examine the substrate." Source: American Museum of Photography http://www.photographymuseum.com/primer.html

Tintype of Two Young Women, Seated, with Man [n.d.]

A small tintype of three unidentified individuals, two women, seated, and a young Black gentleman, standing. The date, location and name of the photographer are unknown. This tintype was in the possession of Iris Sloman Bell, of St. Catharines, Ontario. The Sloman - Bell families have relatives who were former American slaves who came to Canada. They later settled in the London and St. Catharines areas of Ontario."Tintypes were the invention of Prof. Hamilton Smith of Ohio. They begin as thin sheets of iron, covered with a layer of black paint. This serves as the base for the same iodized collodion coating and silver nitrate bath used in the ambrotype process. First made in 1856, millions were produced well into the twentieth century. When tintypes were finished in the same sorts of mats and cases used for ambrotypes, it can be almost impossible to distinguish which process was used without removing the image to examine the substrate." Source: American Museum of Photography http://www.photographymuseum.com/primer.html

Tintype of African American Gentleman Seated on Stool [n.d.]

An unidentified man poses for his portrait seated on a stool in this undated black and white tintype photograph. This tintype was in the possession of Iris Sloman Bell, of St. Catharines, Ontario. Relatives of the Bell - Sloman family include former Black slaves from the United States who settled in Canada."Tintypes were the invention of Prof. Hamilton Smith of Ohio. They begin as thin sheets of iron, covered with a layer of black paint. This serves as the base for the same iodized collodion coating and silver nitrate bath used in the ambrotype process. First made in 1856, millions were produced well into the twentieth century. When tintypes were finished in the same sorts of mats and cases used for ambrotypes, it can be almost impossible to distinguish which process was used without removing the image to examine the substrate." Source: American Museum of Photography http://www.photographymuseum.com/primer.html

Tintype of Man Smoking a Cigar [n.d.]

This small black and white tintype photograph shows a young Black gentleman smoking a cigar. No date or location is provided. This tintype was in the possession of Iris Sloman Bell, of St. Catharines, Ontario. Relatives of the Sloman - Bell families are descended from former slaves from the United States who settled in Canada."Tintypes were the invention of Prof. Hamilton Smith of Ohio. They begin as thin sheets of iron, covered with a layer of black paint. This serves as the base for the same iodized collodion coating and silver nitrate bath used in the ambrotype process. First made in 1856, millions were produced well into the twentieth century. When tintypes were finished in the same sorts of mats and cases used for ambrotypes, it can be almost impossible to distinguish which process was used without removing the image to examine the substrate." Source: American Museum of Photography http://www.photographymuseum.com/primer.html

Tintype of Young African American Woman Seated at Table [n.d.]

Black and white tintype of an unidentified woman seated with hand-colored red detail on her scarf and the tablecloth. The date, location and photographer are unknown. This tintype was in the possession of Iris Sloman Bell, of St. Catharines, Ontario. Relatives of the Bell - Sloman families include former slaves from the United States who escaped to Canada. They later settled in the London and St. Catharines areas of southern Ontario."Tintypes were the invention of Prof. Hamilton Smith of Ohio. They begin as thin sheets of iron, covered with a layer of black paint. This serves as the base for the same iodized collodion coating and silver nitrate bath used in the ambrotype process. First made in 1856, millions were produced well into the twentieth century. When tintypes were finished in the same sorts of mats and cases used for ambrotypes, it can be almost impossible to distinguish which process was used without removing the image to examine the substrate." Source: American Museum of Photography http://www.photographymuseum.com/primer.html

Tintype Portrait of African American Couple [n.d.]

An unidentified couple is pictured in this early black and white tintype photograph. The date, location and name of the photographer are unknown. This tintype was in the possession of Iris Sloman Bell, of St. Catharines, Ontario. The Bell - Sloman families have relatives who are descended from African American slaves who settled in Canada."Tintypes were the invention of Prof. Hamilton Smith of Ohio. They begin as thin sheets of iron, covered with a layer of black paint. This serves as the base for the same iodized collodion coating and silver nitrate bath used in the ambrotype process. First made in 1856, millions were produced well into the twentieth century. When tintypes were finished in the same sorts of mats and cases used for ambrotypes, it can be almost impossible to distinguish which process was used without removing the image to examine the substrate." Source: American Museum of Photography http://www.photographymuseum.com/primer.html

Tintype of Sad Young Black Girl Perched on Bench [n.d.]

This unhappy looking young girl is featured in an early black and white tintype photograph. The date, location and name of the photographer are unknown. This small tintype was in the possession of Iris Sloman Bell, of St. Catharines, Ontario. The Sloman - Bell families have descendants who are former slaves from the United States."Tintypes were the invention of Prof. Hamilton Smith of Ohio. They begin as thin sheets of iron, covered with a layer of black paint. This serves as the base for the same iodized collodion coating and silver nitrate bath used in the ambrotype process. First made in 1856, millions were produced well into the twentieth century. When tintypes were finished in the same sorts of mats and cases used for ambrotypes, it can be almost impossible to distinguish which process was used without removing the image to examine the substrate." Source: American Museum of Photography http://www.photographymuseum.com/primer.html

Small Tintype of Young Black Woman, Seated [n.d.]

A young woman seated with arm resting on a side table is pictured in this small black and white tintype photograph. The date, location and name of the photographer are unknown. This tintype was among the family memorabilia in the possession of Iris Sloman Bell, of St. Catharines, Ontario. The Sloman - Bell families have relatives who were former slaves from the United States. They escaped to Canada and later settled in the London and St. Catharines areas of Ontario."Tintypes were the invention of Prof. Hamilton Smith of Ohio. They begin as thin sheets of iron, covered with a layer of black paint. This serves as the base for the same iodized collodion coating and silver nitrate bath used in the ambrotype process. First made in 1856, millions were produced well into the twentieth century. When tintypes were finished in the same sorts of mats and cases used for ambrotypes, it can be almost impossible to distinguish which process was used without removing the image to examine the substrate." Source: American Museum of Photography http://www.photographymuseum.com/primer.html

Tintype of Girl Peering through Lattice [n.d.]

A young African American girl peers through a lattice window in an unknown photographer's studio in this small black and white tintype. The girl in the photo is unidentified. There are slight scratches on the surface of the tintype, which is cut in a hexagonal shape. This tintype was in the possession of Iris Sloman Bell, of St. Catharines, Ontario. The Sloman - Bell families have relatives who are descended from Black slaves from the United States."Tintypes were the invention of Prof. Hamilton Smith of Ohio. They begin as thin sheets of iron, covered with a layer of black paint. This serves as the base for the same iodized collodion coating and silver nitrate bath used in the ambrotype process. First made in 1856, millions were produced well into the twentieth century. When tintypes were finished in the same sorts of mats and cases used for ambrotypes, it can be almost impossible to distinguish which process was used without removing the image to examine the substrate." Source: American Museum of Photography http://www.photographymuseum.com/primer.html

Tintype of African American Man Standing at Wooden Fence [n.d.]

An unidentified African American man poses beside a wooden fence in the studio of an unknown photographer in this small black and white tintype photograph. A painted landscape serves as the backdrop. This tintype was in the possession of Iris Sloman Bell, of St. Catharines, Ontario. The Sloman - Bell families are descended from former American slaves who settled in Canada."Tintypes were the invention of Prof. Hamilton Smith of Ohio. They begin as thin sheets of iron, covered with a layer of black paint. This serves as the base for the same iodized collodion coating and silver nitrate bath used in the ambrotype process. First made in 1856, millions were produced well into the twentieth century. When tintypes were finished in the same sorts of mats and cases used for ambrotypes, it can be almost impossible to distinguish which process was used without removing the image to examine the substrate." Source: American Museum of Photography http://www.photographymuseum.com/primer.html

Tintype of African American Woman Standing with Book [n.d.]

A woman standing with a book beside a table covered with a decorative cloth is featured in this small black and white tintype photograph. The tintype has been mounted in a paper slip frame with gold edging. There is a small area of red, hand-colored highlighting on the tablecloth. The backdrop appears to be a painted landscape scene. The tintype is undated and there is no indication of the identity of the woman in the photo or the photographer. This tintype was in the possession of Iris Sloman Bell, of St. Catharines, Ontario. The Sloman - Bell families have relatives who are former slaves from the United States who later settled in southern Ontario."Tintypes were the invention of Prof. Hamilton Smith of Ohio. They begin as thin sheets of iron, covered with a layer of black paint. This serves as the base for the same iodized collodion coating and silver nitrate bath used in the ambrotype process. First made in 1856, millions were produced well into the twentieth century. When tintypes were finished in the same sorts of mats and cases used for ambrotypes, it can be almost impossible to distinguish which process was used without removing the image to examine the substrate." Source: American Museum of Photography http://www.photographymuseum.com/primer.html

Tintype of Three African American Women [n.d.]

Three unidentified Black women, two seated and one standing, pose against a painted backdrop in the studio of an unknown photographer. This small black and white tintype has been cut on the upper and lower left-hand corners. The back of the tintype is a copper color. This photograph was among the family memorabilia in the possession of Iris Sloman Bell, of St. Catharines, Ontario. Relatives of the Sloman - Bell families include former American slaves who settled in Canada."Tintypes were the invention of Prof. Hamilton Smith of Ohio. They begin as thin sheets of iron, covered with a layer of black paint. This serves as the base for the same iodized collodion coating and silver nitrate bath used in the ambrotype process. First made in 1856, millions were produced well into the twentieth century. When tintypes were finished in the same sorts of mats and cases used for ambrotypes, it can be almost impossible to distinguish which process was used without removing the image to examine the substrate." Source: American Museum of Photography http://www.photographymuseum.com/primer.html

Tintype of Seated African American Gentleman [n.d.]

An unidentified young African American gentleman sits cross-legged on a decorative wooden stool in this small black and white tintype photograph. The date and name of the photographer are unknown. Hand-colored red detailing is visible on the curtain cord and the tablecloth. This tintype was in the possession of Iris Sloman Bell, of St. Catharines, Ontario. Relatives of the Sloman - Bell families were former American slaves who settled in Canada."Tintypes were the invention of Prof. Hamilton Smith of Ohio. They begin as thin sheets of iron, covered with a layer of black paint. This serves as the base for the same iodized collodion coating and silver nitrate bath used in the ambrotype process. First made in 1856, millions were produced well into the twentieth century. When tintypes were finished in the same sorts of mats and cases used for ambrotypes, it can be almost impossible to distinguish which process was used without removing the image to examine the substrate." Source: American Museum of Photography http://www.photographymuseum.com/primer.html

Tintype of Young African American Woman Standing with Purse [n.d.]

An unidentified young African American woman stands beside a chair in this small black and white tintype, undated. The name of the photographer is unknown. This tintype was in the possession of the Iris Sloman Bell, of St. Catharines. The Sloman - Bell families have relatives who are descended from former American slaves who settled in Canada."Tintypes were the invention of Prof. Hamilton Smith of Ohio. They begin as thin sheets of iron, covered with a layer of black paint. This serves as the base for the same iodized collodion coating and silver nitrate bath used in the ambrotype process. First made in 1856, millions were produced well into the twentieth century. When tintypes were finished in the same sorts of mats and cases used for ambrotypes, it can be almost impossible to distinguish which process was used without removing the image to examine the substrate." Source: American Museum of Photography http://www.photographymuseum.com/primer.html