A detached leaf protocol for rapid screening of germplasm for resistance to citrus canker (Xantho... more A detached leaf protocol for rapid screening of germplasm for resistance to citrus canker (Xanthomonas citri subsp. citri, Xcc) and citrus bacterial spot (Xanthomonas alfalfae subsp. citrumelonis, Xac) was developed to evaluate limited quantities of leaf material. Bacterial inocula of Xcc or Xac at 10 4 , 10 5 , or 10 8 cfu ml −1 were injection-infiltrated into the abaxial surface of disinfested, immature leaves of susceptible and resistant genotypes. Inoculated detached leaves were placed on the surface of 0.5% water agar plates and incubated at 28°C under a 12 h photoperiod. Likewise, inocula were infiltrated into attached leaves of greenhouse plants. At high inoculum concentrations of Xcc or Xac (10 8 cfu ml −1), resistant cultivars of kumquat developed a hypersensitive-like reaction within 3 days post inoculation (dpi). At 10 5 cfu ml −1 , populations 14 dpi were <10 4 per inoculation site. In canker-susceptible Citrus spp. ('Duncan' grapefruit and 'Rough' lemon), watersoaked areas occurred by 3 dpi and typical canker lesions developed by 7 to14 dpi. Concentration of Xcc recovered from inoculation sites was approximately 10 5 cfu ml −1 by 14 dpi. In citrus bacterial spot-susceptible citrus ('Swingle' citrumelo and grapefruit), symptoms developed within 7 dpi. Populations of Xac after inoculation at 10 5 cfu ml −1 were comparable to Xcc in susceptible hosts 14 dpi (>10 5). The detached leaf assay is useful for the characterization and differentiation of lesion phenotype for each Xanthomonas pathogen permitting rapid screening of germplasm resistance based on the quantification of number of lesions and bacterial concentration.
The xylem limited plant pathogenic bacterium Xylella fastidiosa causes economically important dis... more The xylem limited plant pathogenic bacterium Xylella fastidiosa causes economically important diseases on agronomic, horticultural and landscape plants. This review includes the current status of polymerase chain reaction (PCR) based systems for detection and characterization of X. fastidiosa, and presents a genome-wide analysis of strain differentiation. The use of genomics data for strain comparisons will improve the understanding of the genetic determinants of strain specific pathogenicity and virulence. The genome-level analysis can be applied to design new strategies for management and control of Xylella fastidiosa associated diseases in a wide range of crops.
Xylella fastidiosa (25) causes economically important diseases of several agronomic and horticult... more Xylella fastidiosa (25) causes economically important diseases of several agronomic and horticultural crops in North and South America, including alfalfa dwarf, Pierce's disease (PD) of grapevine, citrus variegated chlorosis (CVC), coffee leaf scorch, phony peach, pear leaf scald, pecan leaf scorch, almond leaf scorch, and plum leaf scald (13,23), as well as leaf scorch of several landscape trees and ornamental plants (8). X. fastidiosa-caused diseases
A prototype needle-free device was evaluated for delivery of Xanthomonas citri subsp. citri bacte... more A prototype needle-free device was evaluated for delivery of Xanthomonas citri subsp. citri bacteria into the leaves of cultivars susceptible and resistant to citrus canker. The device delivered a precisely controlled volume of bacterial suspension through infiltration of stomata by injection with pressurized gas. The device produced a uniform inoculation of bacteria into the leaves as measured by the volume of infiltration and diameter of the infiltrated area. No damage to the leaves was observed after inoculation with the automated device, even though a higher number of canker lesions developed compared to a hand-held needleless syringe injection method. The level of practice needed for operation of the automated device was minimal compared to considerable skill required to perform the hand-held injection. Results from inoculations with the automated device are in accord with the results with the hand-held syringe method that demonstrated kumquats are highly resistant to citrus canker while rough lemon and ÔHamlinÕ sweet orange are susceptible.
Synthesis of cDNA probes by random-priming of a viroid template displays the unusual property of ... more Synthesis of cDNA probes by random-priming of a viroid template displays the unusual property of specificity to all members included within a single citrus viroid Group. The specificity of hybridization reactions was influenced by the structural conformation of the viroid RNA template, reaction conditions for reverse transcription and hybridization protocols. Mapping the loci for probe transcription from the CEVd, CVd-IIb, and CVd-IV genomes suggests that a similar structured conformation may be responsible for group specificity. A stem-loop configuration in the viroid template and hybridization target sites can be proposed to be responsible for the availability of the group-specific sequences.
A detached leaf protocol for rapid screening of germplasm for resistance to citrus canker (Xantho... more A detached leaf protocol for rapid screening of germplasm for resistance to citrus canker (Xanthomonas citri subsp. citri, Xcc) and citrus bacterial spot (Xanthomonas alfalfae subsp. citrumelonis, Xac) was developed to evaluate limited quantities of leaf material. Bacterial inocula of Xcc or Xac at 10 4 , 10 5 , or 10 8 cfu ml −1 were injection-infiltrated into the abaxial surface of disinfested, immature leaves of susceptible and resistant genotypes. Inoculated detached leaves were placed on the surface of 0.5% water agar plates and incubated at 28°C under a 12 h photoperiod. Likewise, inocula were infiltrated into attached leaves of greenhouse plants. At high inoculum concentrations of Xcc or Xac (10 8 cfu ml −1), resistant cultivars of kumquat developed a hypersensitive-like reaction within 3 days post inoculation (dpi). At 10 5 cfu ml −1 , populations 14 dpi were <10 4 per inoculation site. In canker-susceptible Citrus spp. ('Duncan' grapefruit and 'Rough' lemon), watersoaked areas occurred by 3 dpi and typical canker lesions developed by 7 to14 dpi. Concentration of Xcc recovered from inoculation sites was approximately 10 5 cfu ml −1 by 14 dpi. In citrus bacterial spot-susceptible citrus ('Swingle' citrumelo and grapefruit), symptoms developed within 7 dpi. Populations of Xac after inoculation at 10 5 cfu ml −1 were comparable to Xcc in susceptible hosts 14 dpi (>10 5). The detached leaf assay is useful for the characterization and differentiation of lesion phenotype for each Xanthomonas pathogen permitting rapid screening of germplasm resistance based on the quantification of number of lesions and bacterial concentration.
CIMMYT® (www.cimmyt.cgiar.org) is an internationally funded, nonprofit scientific research and tr... more CIMMYT® (www.cimmyt.cgiar.org) is an internationally funded, nonprofit scientific research and training organization. Headquartered in Mexico, the Center works with agricultural research institutions worldwide to improve the productivity, profitability, and sustainability of maize and wheat systems for poor farmers in developing countries. It is one of 16 food and environmental centers known as the Future Harvest Centers. The centers, located around the world, conduct research in partnership with farmers, scientists and policymakers to help alleviate poverty and increase food security while protecting natural resources. They are principally funded through the early 60 countries, private foundations, and regional and international organizations that make up the Consultative Group on International Agricultural Research (CGIAR), (www.cgiar.org). Financial support for CIMMYT's research agenda also comes from many other sources, including foundations, development banks, and public and private agencies. In 1998, the centers supported by the CGIAR created Future Harvest, ® a charitable and educational organization that reaches out to media, scholars, and scientists in the world's premier peace, environment, health, population, and development, research organizations, as well as policymakers and civil society. Future Harvest catalyzes action for a world with less poverty, a healthier human family, well-nourished children, and a better environment. It supports research, promotes partnerships, and sponsors on-the-ground projects that bring the results of research to farmers in Africa, Asia and Latin America (see www.futureharvest.org}. The National Agriculture and Livestock Research Institute (I NIA) was created by law 16065 dated October 1989 as a requirement to set up a new research structure for developing appropriate technologies to improve agriculture production and permit the most efficient exploitation of Uruguay's limited land area. INIA is financed by public funds and contributions from farmer's organizations, in equal proportion. Delegates from public sector and farmer's organizations govern INIA through a four-member board. INIA is composed of a network offive regional experimental stations. It has strong relationship with other national and international organizations dedicated to agricultural research and technology transfer. a International Maize and Wheat Improvement Center (CIMMYT) and National Agriculture and Livestock Research Institute (INIA) 2000. All rights reserved. The opinions expressed in this publication are sole responsibility of the authors. The designations employed in the presentation of material in this publication do not imply the expressions of any opinion whatsoever on the part of CIMMYT, INIA, or contributory organizations concerning the legal status of any country, territory, city, or area, or of its authorities, or concerning the delimitation of its frontiers or boundaries. CIMMYT and INIA encourage fair use of this material. Proper citation is requested.
Soil application of the systemic insecticide imidacloprid (Admire®, Bayer Crop Science) produced ... more Soil application of the systemic insecticide imidacloprid (Admire®, Bayer Crop Science) produced season-long control of citrus canker caused by Xanthomonas citri sbsp. citri. Imidacloprid is a neonicotinoid that breaks down in planta into 6chloronicotinic acid, a compound closely related to the systemic acquired resistance (SAR) inducer isonicotinic acid. Potted Swingle citrumelo seedlings (Citrus paradisi × Poncirus trifoliata) were treated with imidacloprid and the SAR inducers, isonicotinic acid, and acibenzolar-s-methyl as soil drenches or with acibenzolar-s-methyl as a foliar spray 1week prior to inoculation of immature leaves with X. citri sbsp. citri. Seedlings were re-inoculated four times over a 24-week period. SAR induction was confirmed by expression of the PR-2 gene (β-1,3 glucanase). Soil drenches of imidacloprid, isonicotinic acid, and acibenzolar-s-methyl induced a high and persistent up-regulation of PR-2 gene expression and reduced the number of canker lesions for up to 24 weeks compared to 4 weeks for foliar acibenzolar-s-methyl. Soil applied inducers of SAR reduced canker lesions up to 70% compared with the untreated inoculated plants. Lesions on leaves were small, necrotic, and flat compared to pustular lesions on inoculated untreated plants. Populations of X. citri sbsp. citri per leaf were reduced 1-3 log units in soil-treated plants compared to inoculated untreated plants. Keywords Gene expression. Pathogenicity-related proteins. Xanthomonas citri Abbreviations ASM acibenzolar-s-methyl Imid imidacloprid INA isonicotinic acid PR-2 pathogenicity-related protein 2 SAR systemic acquired resistance SA salicylic acid Xcc Xanthomonas citri sbsp. citri
The xylem limited plant pathogenic bacterium Xylella fastidiosa causes economically important dis... more The xylem limited plant pathogenic bacterium Xylella fastidiosa causes economically important diseases on agronomic, horticultural and landscape plants. This review includes the current status of polymerase chain reaction (PCR) based systems for detection and characterization of X. fastidiosa, and presents a genome-wide analysis of strain differentiation. The use of genomics data for strain comparisons will improve the understanding of the genetic determinants of strain specific pathogenicity and virulence. The genome-level analysis can be applied to design new strategies for management and control of Xylella fastidiosa associated diseases in a wide range of crops.
Nicotiana tabacum genotype (SR-1), was evaluated as a susceptible host for the bioassay of Xylell... more Nicotiana tabacum genotype (SR-1), was evaluated as a susceptible host for the bioassay of Xylella fastidiosa strains. Readily transformable N. tabacum cv. SR-1 plants were propagated in vitro. Transplanted plants were inoculated with various Xf strains. Inocula consisted of aqueous suspensions of bacterial cells harvested from 7-10 day old cultures on solid PWG medium. Inoculations were made by needle puncture through 20μL of inoculum (108 bacteria/mL) placed in the axils of three basal leaves. Inoculated plants were maintained in a growth room (27-28 o C, 12 hour photoperiod provided by GE High Output fluorescent lights) for 1 month, and subsequently transferred to a greenhouse. Generally, symptoms on plants inoculated with Xf strain Temecula-1 included necrosis at the margins with chlorotic zones extending toward the midvein after 6-8 weeks. Some affected leaves became cupped and curled downward. As infections became systemic, leaves that developed on new shoots were chlorotic an...
A genome-wide search was performed to identify simple sequence repeat (SSR) loci among the availa... more A genome-wide search was performed to identify simple sequence repeat (SSR) loci among the available sequence databases from four strains of Xylella fastidiosa (strains causing Pierce's disease, citrus variegated chlorosis, almond leaf scorch, and oleander leaf scorch). Thirty-four SSR loci were selected for SSR primer design and were validated in PCR experiments. These multilocus SSR primers, distributed across the X. fastidiosa genome, clearly differentiated and clustered X. fastidiosa strains collected from grape, almond, citrus, and oleander. They are well suited for differentiating strains and studying X. fastidiosa epidemiology and population genetics.
Xylella fastidiosa is an important pathogen of many commercial crops. Detection of X. fastidiosa ... more Xylella fastidiosa is an important pathogen of many commercial crops. Detection of X. fastidiosa is difficult due to low concentrations of the bacteria in insects and asymptomatic plant tissue, and non-uniform distribution in infected plants. A dual purpose conventional PCR and quantitative PCR (TaqManä) system was developed for the generic detection of X. fastidiosa strains. Primers HL5 and HL6, designed to amplify a unique region common to the sequenced genomes of four Xylella strains, amplified a 221 bp fragment from strains associated with PierceÕs disease of grapes, almond leaf scorch, and oleander leaf scorch disease and from DNA from an Xf strain associated with citrus variegated chlorosis. Standard curves were obtained using concentrations of Xylella ranging from 5 to 10 5 cells per reaction in water and grape extracts and 10-10 5 cells in insect DNA. Regression curves were similar, with correlation coefficients of r 2 > 0.97. In quantitative PCR, C t values ranged between 20 and 36 cycles for 5-10 5 bacterial cells per reaction. No amplicons were obtained with several non-Xf bacterial strains tested including related plant pathogenic, grape endophytic bacteria and endosymbiotic bacteria isolated from glassy-winged sharpshooters. The method was evaluated for clinical diagnosis of Xf in grapes, almonds and insect vectors. The procedure described is reliable for detection of the pathogen with a high degree of sensitivity and specificity.
Readily transformable Nicotiana tabacum cv. SR1 (Petite Havana) was evaluated as a host for the b... more Readily transformable Nicotiana tabacum cv. SR1 (Petite Havana) was evaluated as a host for the bioassay of Xylella fastidiosa strains. Plant growing conditions and inoculation methods were optimized to enhance symptom expression 4 to 6 weeks post inoculation. Tobacco plants were inoculated with X. fastidiosa strains associated with almond leaf scorch disease (ALSD) and Pierce's disease (PD) of grapevine in California. All PD strains and the ALSD strain Dixon caused characteristic leaf scorch symptoms, whereas two other ALSD-associated strains (M12 and M23) caused severe leaf chlorosis followed by necrosis, leaf death, and drooping of older leaves. Symptoms began to develop 10 to 14 days post inoculation and proceeded to resemble those of X. fastidiosa-infected grape and almond. The presence of X. fastidiosa in affected plants was confirmed by reisolation of the pathogen, enzyme-linked immunosorbent assay, quantitative polymerase chain reaction (QPCR), and observation of X. fast...
The aim of this project is to construct and express in grapevine, a protein or protein chimera ca... more The aim of this project is to construct and express in grapevine, a protein or protein chimera capable of inactivating or otherwise interfering with the infectivity of, or disease-induction by, Xylella fastidiosa (Xf), the causative agent of Pierce's disease (PD) ...
A detached leaf protocol for rapid screening of germplasm for resistance to citrus canker (Xantho... more A detached leaf protocol for rapid screening of germplasm for resistance to citrus canker (Xanthomonas citri subsp. citri, Xcc) and citrus bacterial spot (Xanthomonas alfalfae subsp. citrumelonis, Xac) was developed to evaluate limited quantities of leaf material. Bacterial inocula of Xcc or Xac at 10 4 , 10 5 , or 10 8 cfu ml −1 were injection-infiltrated into the abaxial surface of disinfested, immature leaves of susceptible and resistant genotypes. Inoculated detached leaves were placed on the surface of 0.5% water agar plates and incubated at 28°C under a 12 h photoperiod. Likewise, inocula were infiltrated into attached leaves of greenhouse plants. At high inoculum concentrations of Xcc or Xac (10 8 cfu ml −1), resistant cultivars of kumquat developed a hypersensitive-like reaction within 3 days post inoculation (dpi). At 10 5 cfu ml −1 , populations 14 dpi were <10 4 per inoculation site. In canker-susceptible Citrus spp. ('Duncan' grapefruit and 'Rough' lemon), watersoaked areas occurred by 3 dpi and typical canker lesions developed by 7 to14 dpi. Concentration of Xcc recovered from inoculation sites was approximately 10 5 cfu ml −1 by 14 dpi. In citrus bacterial spot-susceptible citrus ('Swingle' citrumelo and grapefruit), symptoms developed within 7 dpi. Populations of Xac after inoculation at 10 5 cfu ml −1 were comparable to Xcc in susceptible hosts 14 dpi (>10 5). The detached leaf assay is useful for the characterization and differentiation of lesion phenotype for each Xanthomonas pathogen permitting rapid screening of germplasm resistance based on the quantification of number of lesions and bacterial concentration.
The xylem limited plant pathogenic bacterium Xylella fastidiosa causes economically important dis... more The xylem limited plant pathogenic bacterium Xylella fastidiosa causes economically important diseases on agronomic, horticultural and landscape plants. This review includes the current status of polymerase chain reaction (PCR) based systems for detection and characterization of X. fastidiosa, and presents a genome-wide analysis of strain differentiation. The use of genomics data for strain comparisons will improve the understanding of the genetic determinants of strain specific pathogenicity and virulence. The genome-level analysis can be applied to design new strategies for management and control of Xylella fastidiosa associated diseases in a wide range of crops.
Xylella fastidiosa (25) causes economically important diseases of several agronomic and horticult... more Xylella fastidiosa (25) causes economically important diseases of several agronomic and horticultural crops in North and South America, including alfalfa dwarf, Pierce's disease (PD) of grapevine, citrus variegated chlorosis (CVC), coffee leaf scorch, phony peach, pear leaf scald, pecan leaf scorch, almond leaf scorch, and plum leaf scald (13,23), as well as leaf scorch of several landscape trees and ornamental plants (8). X. fastidiosa-caused diseases
A prototype needle-free device was evaluated for delivery of Xanthomonas citri subsp. citri bacte... more A prototype needle-free device was evaluated for delivery of Xanthomonas citri subsp. citri bacteria into the leaves of cultivars susceptible and resistant to citrus canker. The device delivered a precisely controlled volume of bacterial suspension through infiltration of stomata by injection with pressurized gas. The device produced a uniform inoculation of bacteria into the leaves as measured by the volume of infiltration and diameter of the infiltrated area. No damage to the leaves was observed after inoculation with the automated device, even though a higher number of canker lesions developed compared to a hand-held needleless syringe injection method. The level of practice needed for operation of the automated device was minimal compared to considerable skill required to perform the hand-held injection. Results from inoculations with the automated device are in accord with the results with the hand-held syringe method that demonstrated kumquats are highly resistant to citrus canker while rough lemon and ÔHamlinÕ sweet orange are susceptible.
Synthesis of cDNA probes by random-priming of a viroid template displays the unusual property of ... more Synthesis of cDNA probes by random-priming of a viroid template displays the unusual property of specificity to all members included within a single citrus viroid Group. The specificity of hybridization reactions was influenced by the structural conformation of the viroid RNA template, reaction conditions for reverse transcription and hybridization protocols. Mapping the loci for probe transcription from the CEVd, CVd-IIb, and CVd-IV genomes suggests that a similar structured conformation may be responsible for group specificity. A stem-loop configuration in the viroid template and hybridization target sites can be proposed to be responsible for the availability of the group-specific sequences.
A detached leaf protocol for rapid screening of germplasm for resistance to citrus canker (Xantho... more A detached leaf protocol for rapid screening of germplasm for resistance to citrus canker (Xanthomonas citri subsp. citri, Xcc) and citrus bacterial spot (Xanthomonas alfalfae subsp. citrumelonis, Xac) was developed to evaluate limited quantities of leaf material. Bacterial inocula of Xcc or Xac at 10 4 , 10 5 , or 10 8 cfu ml −1 were injection-infiltrated into the abaxial surface of disinfested, immature leaves of susceptible and resistant genotypes. Inoculated detached leaves were placed on the surface of 0.5% water agar plates and incubated at 28°C under a 12 h photoperiod. Likewise, inocula were infiltrated into attached leaves of greenhouse plants. At high inoculum concentrations of Xcc or Xac (10 8 cfu ml −1), resistant cultivars of kumquat developed a hypersensitive-like reaction within 3 days post inoculation (dpi). At 10 5 cfu ml −1 , populations 14 dpi were <10 4 per inoculation site. In canker-susceptible Citrus spp. ('Duncan' grapefruit and 'Rough' lemon), watersoaked areas occurred by 3 dpi and typical canker lesions developed by 7 to14 dpi. Concentration of Xcc recovered from inoculation sites was approximately 10 5 cfu ml −1 by 14 dpi. In citrus bacterial spot-susceptible citrus ('Swingle' citrumelo and grapefruit), symptoms developed within 7 dpi. Populations of Xac after inoculation at 10 5 cfu ml −1 were comparable to Xcc in susceptible hosts 14 dpi (>10 5). The detached leaf assay is useful for the characterization and differentiation of lesion phenotype for each Xanthomonas pathogen permitting rapid screening of germplasm resistance based on the quantification of number of lesions and bacterial concentration.
CIMMYT® (www.cimmyt.cgiar.org) is an internationally funded, nonprofit scientific research and tr... more CIMMYT® (www.cimmyt.cgiar.org) is an internationally funded, nonprofit scientific research and training organization. Headquartered in Mexico, the Center works with agricultural research institutions worldwide to improve the productivity, profitability, and sustainability of maize and wheat systems for poor farmers in developing countries. It is one of 16 food and environmental centers known as the Future Harvest Centers. The centers, located around the world, conduct research in partnership with farmers, scientists and policymakers to help alleviate poverty and increase food security while protecting natural resources. They are principally funded through the early 60 countries, private foundations, and regional and international organizations that make up the Consultative Group on International Agricultural Research (CGIAR), (www.cgiar.org). Financial support for CIMMYT's research agenda also comes from many other sources, including foundations, development banks, and public and private agencies. In 1998, the centers supported by the CGIAR created Future Harvest, ® a charitable and educational organization that reaches out to media, scholars, and scientists in the world's premier peace, environment, health, population, and development, research organizations, as well as policymakers and civil society. Future Harvest catalyzes action for a world with less poverty, a healthier human family, well-nourished children, and a better environment. It supports research, promotes partnerships, and sponsors on-the-ground projects that bring the results of research to farmers in Africa, Asia and Latin America (see www.futureharvest.org}. The National Agriculture and Livestock Research Institute (I NIA) was created by law 16065 dated October 1989 as a requirement to set up a new research structure for developing appropriate technologies to improve agriculture production and permit the most efficient exploitation of Uruguay's limited land area. INIA is financed by public funds and contributions from farmer's organizations, in equal proportion. Delegates from public sector and farmer's organizations govern INIA through a four-member board. INIA is composed of a network offive regional experimental stations. It has strong relationship with other national and international organizations dedicated to agricultural research and technology transfer. a International Maize and Wheat Improvement Center (CIMMYT) and National Agriculture and Livestock Research Institute (INIA) 2000. All rights reserved. The opinions expressed in this publication are sole responsibility of the authors. The designations employed in the presentation of material in this publication do not imply the expressions of any opinion whatsoever on the part of CIMMYT, INIA, or contributory organizations concerning the legal status of any country, territory, city, or area, or of its authorities, or concerning the delimitation of its frontiers or boundaries. CIMMYT and INIA encourage fair use of this material. Proper citation is requested.
Soil application of the systemic insecticide imidacloprid (Admire®, Bayer Crop Science) produced ... more Soil application of the systemic insecticide imidacloprid (Admire®, Bayer Crop Science) produced season-long control of citrus canker caused by Xanthomonas citri sbsp. citri. Imidacloprid is a neonicotinoid that breaks down in planta into 6chloronicotinic acid, a compound closely related to the systemic acquired resistance (SAR) inducer isonicotinic acid. Potted Swingle citrumelo seedlings (Citrus paradisi × Poncirus trifoliata) were treated with imidacloprid and the SAR inducers, isonicotinic acid, and acibenzolar-s-methyl as soil drenches or with acibenzolar-s-methyl as a foliar spray 1week prior to inoculation of immature leaves with X. citri sbsp. citri. Seedlings were re-inoculated four times over a 24-week period. SAR induction was confirmed by expression of the PR-2 gene (β-1,3 glucanase). Soil drenches of imidacloprid, isonicotinic acid, and acibenzolar-s-methyl induced a high and persistent up-regulation of PR-2 gene expression and reduced the number of canker lesions for up to 24 weeks compared to 4 weeks for foliar acibenzolar-s-methyl. Soil applied inducers of SAR reduced canker lesions up to 70% compared with the untreated inoculated plants. Lesions on leaves were small, necrotic, and flat compared to pustular lesions on inoculated untreated plants. Populations of X. citri sbsp. citri per leaf were reduced 1-3 log units in soil-treated plants compared to inoculated untreated plants. Keywords Gene expression. Pathogenicity-related proteins. Xanthomonas citri Abbreviations ASM acibenzolar-s-methyl Imid imidacloprid INA isonicotinic acid PR-2 pathogenicity-related protein 2 SAR systemic acquired resistance SA salicylic acid Xcc Xanthomonas citri sbsp. citri
The xylem limited plant pathogenic bacterium Xylella fastidiosa causes economically important dis... more The xylem limited plant pathogenic bacterium Xylella fastidiosa causes economically important diseases on agronomic, horticultural and landscape plants. This review includes the current status of polymerase chain reaction (PCR) based systems for detection and characterization of X. fastidiosa, and presents a genome-wide analysis of strain differentiation. The use of genomics data for strain comparisons will improve the understanding of the genetic determinants of strain specific pathogenicity and virulence. The genome-level analysis can be applied to design new strategies for management and control of Xylella fastidiosa associated diseases in a wide range of crops.
Nicotiana tabacum genotype (SR-1), was evaluated as a susceptible host for the bioassay of Xylell... more Nicotiana tabacum genotype (SR-1), was evaluated as a susceptible host for the bioassay of Xylella fastidiosa strains. Readily transformable N. tabacum cv. SR-1 plants were propagated in vitro. Transplanted plants were inoculated with various Xf strains. Inocula consisted of aqueous suspensions of bacterial cells harvested from 7-10 day old cultures on solid PWG medium. Inoculations were made by needle puncture through 20μL of inoculum (108 bacteria/mL) placed in the axils of three basal leaves. Inoculated plants were maintained in a growth room (27-28 o C, 12 hour photoperiod provided by GE High Output fluorescent lights) for 1 month, and subsequently transferred to a greenhouse. Generally, symptoms on plants inoculated with Xf strain Temecula-1 included necrosis at the margins with chlorotic zones extending toward the midvein after 6-8 weeks. Some affected leaves became cupped and curled downward. As infections became systemic, leaves that developed on new shoots were chlorotic an...
A genome-wide search was performed to identify simple sequence repeat (SSR) loci among the availa... more A genome-wide search was performed to identify simple sequence repeat (SSR) loci among the available sequence databases from four strains of Xylella fastidiosa (strains causing Pierce's disease, citrus variegated chlorosis, almond leaf scorch, and oleander leaf scorch). Thirty-four SSR loci were selected for SSR primer design and were validated in PCR experiments. These multilocus SSR primers, distributed across the X. fastidiosa genome, clearly differentiated and clustered X. fastidiosa strains collected from grape, almond, citrus, and oleander. They are well suited for differentiating strains and studying X. fastidiosa epidemiology and population genetics.
Xylella fastidiosa is an important pathogen of many commercial crops. Detection of X. fastidiosa ... more Xylella fastidiosa is an important pathogen of many commercial crops. Detection of X. fastidiosa is difficult due to low concentrations of the bacteria in insects and asymptomatic plant tissue, and non-uniform distribution in infected plants. A dual purpose conventional PCR and quantitative PCR (TaqManä) system was developed for the generic detection of X. fastidiosa strains. Primers HL5 and HL6, designed to amplify a unique region common to the sequenced genomes of four Xylella strains, amplified a 221 bp fragment from strains associated with PierceÕs disease of grapes, almond leaf scorch, and oleander leaf scorch disease and from DNA from an Xf strain associated with citrus variegated chlorosis. Standard curves were obtained using concentrations of Xylella ranging from 5 to 10 5 cells per reaction in water and grape extracts and 10-10 5 cells in insect DNA. Regression curves were similar, with correlation coefficients of r 2 > 0.97. In quantitative PCR, C t values ranged between 20 and 36 cycles for 5-10 5 bacterial cells per reaction. No amplicons were obtained with several non-Xf bacterial strains tested including related plant pathogenic, grape endophytic bacteria and endosymbiotic bacteria isolated from glassy-winged sharpshooters. The method was evaluated for clinical diagnosis of Xf in grapes, almonds and insect vectors. The procedure described is reliable for detection of the pathogen with a high degree of sensitivity and specificity.
Readily transformable Nicotiana tabacum cv. SR1 (Petite Havana) was evaluated as a host for the b... more Readily transformable Nicotiana tabacum cv. SR1 (Petite Havana) was evaluated as a host for the bioassay of Xylella fastidiosa strains. Plant growing conditions and inoculation methods were optimized to enhance symptom expression 4 to 6 weeks post inoculation. Tobacco plants were inoculated with X. fastidiosa strains associated with almond leaf scorch disease (ALSD) and Pierce's disease (PD) of grapevine in California. All PD strains and the ALSD strain Dixon caused characteristic leaf scorch symptoms, whereas two other ALSD-associated strains (M12 and M23) caused severe leaf chlorosis followed by necrosis, leaf death, and drooping of older leaves. Symptoms began to develop 10 to 14 days post inoculation and proceeded to resemble those of X. fastidiosa-infected grape and almond. The presence of X. fastidiosa in affected plants was confirmed by reisolation of the pathogen, enzyme-linked immunosorbent assay, quantitative polymerase chain reaction (QPCR), and observation of X. fast...
The aim of this project is to construct and express in grapevine, a protein or protein chimera ca... more The aim of this project is to construct and express in grapevine, a protein or protein chimera capable of inactivating or otherwise interfering with the infectivity of, or disease-induction by, Xylella fastidiosa (Xf), the causative agent of Pierce's disease (PD) ...
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