scholarly journals Strain-specific detection of two Aureobasidium pullulans strains, fungal biocontrol agents of fire blight by new, developed multiplex-PCR

2008 ◽  
Vol 104 (5) ◽  
pp. 1433-1441 ◽  
Author(s):  
I. Loncaric ◽  
C. Donat ◽  
B. Antlinger ◽  
J.T. Oberlerchner ◽  
B. Heissenberger ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Aureobasidium Pullulans ◽  
Fire Blight ◽  
Biocontrol Agents ◽  
Specific Detection

scholarly journals A Multiplex-PCR Method for Strain Identification and Detailed Phylogenetic Analysis of AY-Group Phytoplasmas

Plant Disease ◽  
2014 ◽  
Vol 98 (3) ◽  
pp. 299-305 ◽  
Author(s):  
Shigeyuki Kakizawa ◽  
Yoichi Kamagata
Keyword(s):  
Phylogenetic Analysis ◽  
Multiplex Pcr ◽  
16S Rdna ◽  
Pathogenic Bacteria ◽  
Target Genes ◽  
Direct Sequencing ◽  
Pcr Amplification ◽  
Single Copy ◽  
Strain Identification ◽  
Specific Detection

Phytoplasmas are plant pathogenic bacteria that cause devastating losses in the yield of diverse crops worldwide. Specific detection and strain identification of phytoplasmas is important to prevent the spread of phytoplasma-induced diseases. Hence, methods to rapidly detect these organisms are important for pest control. Polymerase chain reaction (PCR) methods using phytoplasma-specific primers are widely used to detect phytoplasmas from infected plants and insects because they are highly sensitive, easily handled, and have a variety of analytical secondary applications. The phytoplasma 16S rDNA was widely used as a target of the PCR detection method; however, further target genes and more rapid methods have been required for more specific detection of phytoplasmas. Here, we developed a multiplex-PCR system to amplify several phytoplasma genes. We designed 36 primers, based on the genome sequence of ‘Candidatus Phytoplasma asteris’, to amplify 18 single-copy genes covering wide regions of the phytoplasma genome. Nine genes could be simultaneously amplified in a single PCR. This multiplex-PCR was applied to DNAs from 10 phytoplasma strains belonging to the AY-group, and different amplification patterns were obtained between strains, suggesting that this method would allow us to differentiate phytoplasmas at the strain level. Direct sequencing was also possible after the multiplex-PCR amplification by a modified sequencing method. Detailed phylogenetic analysis was performed using concatenated sequences, and evolutionary relationships among four Japanese isolates were revealed, where these strains could not be distinguished by their 16S rDNA. Thus, this multiplex-PCR system is useful for rapid strain identification and detailed phylogenetic analysis of phytoplasmas.

scholarly journals Crab Apple Blossoms as a Model for Research on Biological Control of Fire Blight

1997 ◽  
Vol 87 (11) ◽  
pp. 1096-1102 ◽  
Author(s):  
P. L. Pusey
Keyword(s):  
Fire Blight ◽  
Biocontrol Agents ◽  
Suitable Model ◽  
Bacterial Strains ◽  
Bacterial Populations ◽  
Cold Room ◽  
Series Of Experiments ◽  
Population Interactions ◽  
Intact Crab ◽  
Crab Apple

Nonseasonal availability of pomaceous flowers could improve laboratory detection and prefield testing of biocontrol agents for fire blight of pear and apple. Crab apple was selected as a model because of its high flower productivity on 1-year-old wood, high susceptibility to fire blight, and availability from nurseries. Cultivars Manchurian and Snowdrift were manipulated to bloom once by transferring dormant nursery trees from a cold room to a greenhouse and a second time by defoliating trees and applying 1% cytokinin and 0.1% gibberellins to the buds with a brush. Different sets of trees were induced at different times to bloom, so that flowers were produced 12 months in the year. When known bacterial antagonists (Erwinia herbicola strain C9-1 and Pseudomonas fluorescens strain A506) were applied alone or in combination to the stigmas of detached crab apple blossoms prior to inoculation with the pathogen (E. amylovora strain Ea153), population interactions over time were comparable to those reported in previous studies involving pear or apple. In a subsequent series of experiments, the relative effects of 12 bacterial strains on stigmatic populations of strain Ea153 were similar for detached blossoms of crab apple in the laboratory, blossoms of intact crab apple trees in the greenhouse, and blossoms of pear and apple in the field. Additionally, when stigmas of detached crab apple blossoms were inoculated with antagonists (strains C9-1 and A506) and the pathogen, and later subjected to a 24-h wetting period, bacterial populations in the flower hypanthium increased and disease was suppressed. These studies indicate that crab apple blossoms can serve as a suitable model for year-round evaluation and study of biocontrol agents for fire blight.

scholarly journals Bee Vectoring: Development of the Japanese Orchard Bee as a Targeted Delivery System of Biological Control Agents for Fire Blight Management

Pathogens ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 41 ◽  
Author(s):  
Neelendra K. Joshi ◽  
Henry K. Ngugi ◽  
David J. Biddinger
Keyword(s):  
Biological Control ◽  
Targeted Delivery ◽  
Fire Blight ◽  
Erwinia Amylovora ◽  
The United States ◽  
Biocontrol Agents ◽  
Efficient System ◽  
Control Product ◽  
Tree Fruits ◽  
Potential Use

Fire blight, which is caused by the bacteria Erwinia amylovora, remains one of the most important diseases limiting the productivity of apple and pear orchards in the United States. In commercial orchards, in-season fire blight management relies exclusively on the use of antibiotic treatments (such as streptomycin and oxytetracycline) and on bacterial biocontrol agents whose efficacy is limited. We hypothesize that the efficacy of the biocontrol agents can be greatly enhanced through targeted delivery to flowers, which serve as initial infection courts, using the Japanese orchard bee, Osmia cornifrons. Many factors, such as the synchrony of life cycle with plant phenology and specificity to pomaceous plants, suggest that O. cornifrons could be an excellent vector of the biocontrol products during bloom in pome tree fruits. However, deployment of this pollinator species to deliver biocontrol agents for fire blight control has not been attempted previously due to the lack of an efficient system to pack the bodies of the bees exiting nesting tubes with the biocontrol products. In this study, we design and test a dispenser system to facilitate the use of O. conifrons as a vector for commercially available biocontrol products for fire blight control. The effectiveness of O. conifrons to deliver biocontrol agents to flowers, and to effect secondary dissemination from treated to untreated flowers is also evaluated in greenhouse experiments. We found that the O. conifrons bees were able to use the nest dispenser designed for the delivery of biological control products, and are effective in vectoring and delivering the Bacillus subtilis-based biological control product (Serenade®) to apple blossoms. We also found that the O. cornifrons were effective in secondary inoculation of this biological control product to newly-opened flowers. These findings suggest the potential use of commercially available O. conifrons and other orchard bees in targeted delivery of biological control products for fire blight, and possibly other diseases, in different fruit crops.

CHARACTERISATION OF TWO FLUORESCENT STRAINS OF PSEUDOMONAS AS BIOCONTROL AGENTS AGAINST FIRE BLIGHT

2002 ◽  
pp. 299-307 ◽  
Author(s):  
O. Galasso ◽  
G. Sponza ◽  
C. Bazzi ◽  
J.L. Vanneste
Keyword(s):  
Fire Blight ◽  
Biocontrol Agents

scholarly journals Detection and Diversity Assessment of Xylella fastidiosa in Field-Collected Plant and Insect Samples by Using 16S rRNA and gyrB Sequences

2003 ◽  
Vol 69 (7) ◽  
pp. 4249-4255 ◽  
Author(s):  
Jorge L. M. Rodrigues ◽  
M. E. Silva-Stenico ◽  
J. E. Gomes ◽  
J. R. S. Lopes ◽  
S. M. Tsai
Keyword(s):  
16S Rrna ◽  
Multiplex Pcr ◽  
Xylella Fastidiosa ◽  
Rrna Gene ◽  
Specific Detection ◽  
Disease Vector ◽  
Diversity Assessment ◽  
Pcr Products ◽  
Pure Cultures ◽  
The 16S Rrna Gene

ABSTRACT The causal agent of diseases in many economically important plants is attributed to the xylem-limited bacterium Xylella fastidiosa. The detection of this plant pathogen has been hampered due to its difficult isolation and slow growth on plates. Nearly complete nucleotide sequences of the 16S rRNA gene and partial sequences of the gyrB gene were determined for 18 strains of X. fastidiosa isolated from different plant hosts. A phylogenetic analysis, based on gyrB, grouped strains in three clusters; grape-isolated strains formed one cluster, citrus-coffee strains formed another cluster, and a third cluster resulted from all other strains. Primer pairs designed for the 16S rRNA and gyrB genes were extensively searched in databases to verify their in silico specificity. Primer pairs were certified with 30 target and 36 nontarget pure cultures of microorganisms, confirming 100% specificity. A multiplex PCR protocol was developed and its sensitivity tested. Sequencing of PCR products confirmed the validity of the multiplex PCR. Xylella fastidiosa was detected in field-collected plants, disease vector insects, and nonsymptomatic but infected plants. Specific detection of X. fastidiosa may facilitate the understanding of its ecological significance and prevention of spread of the disease.

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