Progress 01/01/11 to 12/31/11
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? 1. Presentations. PI Nunney gave three major presentations. In August, he gave a presentation at the annual American Phytopathological Society meeting in Honolulu “Homologous recombination and the invasion of a new plant host by the pathogenic bacterium, Xylella fastidiosa” and in November he gave an invited talk at UC Riverside (Entomology Dept.) “The role of intercontinental homologous recombination in the complex evolution of Xylella fastidiosa”. In December, he gave an invited presentation at the annual Pierce’s Disease Research Symposium in Sacramento “Homologous recombination and the invasion of a new plant host by Xylella fastidiosa” 2. Websites. We continue to maintain and update two websites to provide information about Xylella and the plant diseases that it causes, to introduce the goals of the project, and to solicit samples from plants exhibiting suspected Xylella infection. The first site (www.xylella.org) is our primary website for agricultural professionals to report leaf-scorch disease outbreaks, to which we will respond with sampling kits. It also includes an interactive map so that users can identify locations of isolates previously documented on specific plant hosts. Collaborator L. Morano (Univ. Houston, Downtown) has established a second site: (www.uhd.edu/academic/colleges/sciences/ns/pdresearch/index.html) which is being used for outreach to growers in Texas, where the Pierce’s disease of grape is a major concern. 3. Genetic Data. We have significantly enlarged the genetic information available in our public MLST database at pubmlst.org/xfastidiosa. The data will continue to be increased as our analyses of the various forms of Xylella are published. All sequence data referenced in publications are also available from Genbank. 4. Data Correction. We have identified and communicated to the research community serious problems concerning two important Xylella resources: first, that the culture collection ATCC was providing the wrong genotype (in fact the wrong subspecies) in place of a strain commonly used in experimental work (Temecula-1); and second, that the draft genome of the strain Ann1, previously used in several research publications, was actually a mix of two different subspecies. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During this year we have continued to work on developing a rigorous framework for monitoring Xylella. This effort has focused on documenting genetic variation, providing an understanding of the origins of this variation, and how it is linked to the infection of different plant hosts. We have now completed multilocus sequence typing (MLST) on almost 400 isolates from the US, Brazil, and Costa Rica. We have recently written a paper detailing the importance of MLST in microbiological studies such as this (Nunney et al. 2012). Last year we concluded that subspp. fastidiosa and sandyi were introduced into the US. In particular, subsp. fastidiosa, that causes an economically damaging disease of grape (Pierce's disease, PD), had been assumed to be native; however, our data are consistent with a single genotype being introduced from Central America around 150 years ago (Nunney et al 2010; Yuan et al 2010). This year (2011) our focus has been on the other two subspecies, subsp. multiplex, that we conclude is native to the US, and subsp. pauca, that is restricted to S. America. We have typed 112 isolates of subsp. multiplex and shown that different MLST sequence types (STs) show strong plant-host associations (Nunney et al, draft MS). We completed our analysis of subsp. pauca after typing 78 isolates from Brazil. This subspecies causes citrus variegated chlorosis (CVC), a serious disease of citrus, and is considered a major biosecurity threat to the US (it is listed as a USDA select agent). Our most important conclusion is that the form of subsp. pauca causing CVC is in fact an inter-subspecific recombinant between subspp. pauca and multiplex. We proposed that this mixing was made possible by the relatively recent introduction of subsp. multiplex into South America, probably in the 1970s (Nunney et al, Submitted). This example highlights our thesis that inter-subspecific recombination is an important mechanism leading to the infection of novel hosts. This view is further supported by our analysis of mulberry-type isolates in the US. Our MLST of 17 mulberry types from across the US (DC to CA) showed them to be (a) genetically homogeneous but (b) a recombinant mix of two subspecies, fastidiosa and multiplex. To confirm this view we sequenced the complete genome of one mulberry type. Analysis is not yet complete but our initial estimate is that it took at least 200 homologous recombination events to create this genome. No other forms of Xylella have been isolated from mulberry. Our experimental study of the plant-host specificity of the different subspecies and MLST sequence types within subspecies has been ongoing for 5 years at two locations (Apopka, FL and Riverside, CA) across a standard set of plant hosts. Unlike previous studies, we can test for variation in infection due to geographical location and year-to-year differences. Our general conclusions are that some plant hosts may be driving specialization (e.g. grape, oleander, mulberry, peach, citrus), while others may permit mixing of the different genotypes (almond, plum). The first of a series of manuscripts detailing this work is currently being written.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2012
Citation:
Nunney, L., S. Elfekih and R. Stouthamer. 2012. The importance of multilocus sequence typing: cautionary tales from the
bacterium Xylella fastidiosa. Phytopath. (In Press).
- Type:
Journal Articles
Status:
Submitted
Year Published:
2012
Citation:
Nunney,, L., X. Yuan, R. E. Bromley, and R. Stouthamer 2012. Inter-subspecific recombination and host specificity of Xylella
fastidiosa in Brazil. Appl. Exp. Microbiol. (Re-submitted following revision).
|
Progress 01/01/07 to 12/31/11
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? 1. Presentations. (a) Talks at the annual American Phytopathological Society meetings by PI Nunney: 07/2008; 07/2009 (2 talks); 07/2011. (b) Talks & posters at the annual Pierce’s Disease Research Symposium: PI Nunney gave invited talks in 12/2007 and 12/2011; the research group presented posters 12/2008, 12/2009 (2 posters), and 12/1010 (3 posters). (c) Talk at the joint annual “Evolution” meeting of the national societies Society for the Study of Evolution, American Society of Naturalists, and Society of Systematic Biologists (06/2009). (d) Invited presentations by PI Nunney at USDA Parlier (4/10), the Biology Department UC Riverside (4/10) and the Entomology Dept UC Riverside (11/11). PI Nunney has been invited to be the keynote speaker at the next Mexican Plant Pathology meeting (07/2013). (e) Joint UC Riverside/Los Alamos National Lab Biosecurity Group Meetings. PI Nunney gave talks in 09/2008 and 05/2010. 2. Websites. We developed and maintained the website www.xylella-fastidiosa.org with the following features: (a) It introduces the goals of the project and lists the four subspecies of X. fastidiosa and their known plant hosts. (b) It has a library of photographs illustrating disease symptoms on plant species. (c) It allows agricultural professionals to report leaf-scorch disease outbreaks and tells them how to submit samples from plants exhibiting suspected Xylella infection. (d) It includes an interactive map (searchable by subspecies and plant host) so that users can identify locations of specific isolates on specific plant hosts. Collaborator L. Morano (Univ. Houston, Downtown) has established a second site, (www.uhd.edu/academic/colleges/sciences/ns/pdresearch/index.html), which is being used for outreach to growers in Texas, where the Pierce’s disease of grape is a major concern. 3. Database. We have established a X. fastidiosa database for multilocus sequence typing (MLST) information within the public MLST site pubmlst.org. We have submitted data on 250 isolates that is freely available to the public. All sequence data has also been submitted to GenBank. 4. Data Correction. We have identified and communicated to the research community serious problems concerning two important Xylella resources: first, that the major national culture collection (ATCC) was providing the wrong genotype (in fact the wrong subspecies) when researchers requested Temecula-1, a strain commonly used in experimental work; and second, that the draft genome of the strain Ann1, previously used in several research publications, was actually a mix of two different subspecies (see Nunney et al 2012a). 5. Statistical Tests. We developed “the introgression test”, a new statistical method for detecting genetic exchange between genetically divergent populations (see Nunney et al 2012b). A program for evaluating the statistical significance of regions of suspected DNA sequence introgression is available from PI Nunney. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We made important discoveries on the roles of biogeography and intersubspecific homologous recombination (IHR) in X. fastidiosa that have broad implications for biosecurity. In particular, we documented the role of IHR in the invasion of new plant hosts. We first established a robust multilocus typing (MLST) for X. fastidiosa using sequence from 7 housekeeping loci and used it to show that 2 of the 3 subspecies found in the US, fastidiosa and sandyi, exhibit the very low levels of genetic variation consistent with an introduction (Yuan et al 2010). We also documented introgression from the native subspecies, multiplex, into subsp. fastidiosa. Subsp. fastidiosa causes Pierce’s disease in grapevine and we established that this subspecies is non-native to the US, having been introduced from Central America as a single isolate, probably in the mid 1800s on a coffee plant shipped to California (Nunney et al 2010). Analysis of 2 subsp. fastidiosa and 1 subsp. multiplex genomes indicated that the subsp. fastidiosa within the US have diversified from the single ancestor primarily via IHR with subsp. multiplex. Subsp. multiplex is native to the US and underwent a recent radiation to produce an array of relatively host-specific genotypes (Nunney et al 2013a). Within this subspecies a distinct group exhibits high levels of IHR, all sharing the same subsp. fastidiosa donor, a donor no longer found in N. America (Nunney et al 2013b). Some of these IHR types have invaded a new host (blueberry) and a second that may be new (blackberry). We found an even more extreme example of an IHR-induced host shift, the shift to mulberry by a genotype that is a roughly equal mix of subspp. multiplex and fastidiosa (Nunney et al 2013c). Citrus variegated chlorosis (CVC) is an economically damaging disease of citrus in Brazil (but absent from the US) caused by subsp. pauca. Using MLST we found that isolates from citrus (and coffee) show little genetic variation but substantial evidence of IHR (Nunney et al 2012b). Using a new statistical test, we found high levels of introgression that suggests why infection of these plants was very recent. We proposed that truly native subsp. pauca (not yet isolated) does not cause disease in citrus or coffee, but that IHR with a novel form of X. fastidiosa (possibly introduced subsp. multiplex) provided the required genetic variation. The project used MLST and genomic data. Yuan et al (2010) showed MLST was more effective at typing at the species/subspecies level and at detecting recombination than 3 other commonly used genetic typing methods, and Nunney et al (2012a) argued that even in the age of genomics MLST provides the rapid and reliable typing that is critical in the interpretation of experimental results (such as variation in host range), and for ensuring that isolates are not contaminated. We identified 2 examples of contamination (see above) that have had serious adverse effects on X. fastidiosa research. We conducted a series of experiments over 4 years documenting host range of different X. fastidiosa genotypes using a panel of plant hosts. These data will be written up for publication in the near future.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Nunney, L., D. Vickerman, R. E. Bromley, S. Russell, J. Hartman, L. D. Morano, and R. Stouthamer. 2013a. Recent radiation and host plant specialization in Xylella fastidiosa native to the United States. Appl. Environ. Microbiol. 79: 2189-2200.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Nunney, L., X. Yuan, R. E. Bromley, and R. Stouthamer. 2012b. Detecting genetic introgression: high levels of inter-subspecific recombination found in Xylella fastidiosa in Brazil. Appl. Environ. Microbiol. 78: 4702-4714.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Nunney, L., S. Elfekih and R. Stouthamer. 2012a. The importance of multilocus sequence typing: cautionary tales from the bacterium Xylella fastidiosa. Phytopath. 102: 456-460.
- Type:
Journal Articles
Status:
Published
Year Published:
2010
Citation:
Nunney, L. , X. Yuan, R. Bromley, J. Hartung, M. Montero-Astua, L. Moreira, B. Ortiz, R. Stouthamer. 2010 Population genomic analysis of a bacterial plant pathogen: novel insight into the origins of Pierces disease of grapevine in the U.S. PLoS ONE 5: e15488 (9pp).
- Type:
Journal Articles
Status:
Other
Year Published:
2013
Citation:
Nunney.L., E. L. Schuenzel, M. Scally, R. E. Bromley, and R. Stouthamer. 2013c. Large scale intersubspecific recombination in the plant pathogenic bacterium Xylella fastidiosa is associated with the host shift to mulberry (In draft).
- Type:
Journal Articles
Status:
Under Review
Year Published:
2013
Citation:
Nunney, L., D. Hopkins, L. D. Morano, S. Russell, and R. Stouthamer. 2013b. Intersubspecific recombination in Xylella fastidiosa native to the United States: infection of a novel host results from an unsuccessful invasion. Appl. Environ. Microbiol. (Submitted)
- Type:
Journal Articles
Status:
Published
Year Published:
2010
Citation:
Yuan, X., L. B. Morano, R. Bromley, S, Spring-Pearson, R. Stouthamer, and L. Nunney 2010 Multilocus sequence typing of Xylella fastidiosa causing Pierce's disease and oleander leaf scorch in the United States. Phytopathology 100: 601-611.
|
Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: 1. Presentations. PI Nunney gave three major presentations. In August, he gave a presentation at the annual American Phytopathological Society meeting in Honolulu "Homologous recombination and the invasion of a new plant host by the pathogenic bacterium, Xylella fastidiosa" and in November he gave an invited talk at UC Riverside (Entomology Dept.) "The role of inter-continental homologous recombination in the complex evolution of Xylella fastidiosa". In December, he gave an invited presentation at the annual Pierce's Disease Research Symposium in Sacramento "Homologous recombination and the invasion of a new plant host by Xylella fastidiosa" 2. Websites. We continue to maintain and update two websites to provide information about Xylella and the plant diseases that it causes, to introduce the goals of the project, and to solicit samples from plants exhibiting suspected Xylella infection. The first site (www.xylella.org) is our primary website for agricultural professionals to report leaf-scorch disease outbreaks, to which we will respond with sampling kits. It also includes an interactive map so that users can identify locations of isolates previously documented on specific plant hosts. Collaborator L. Morano (Univ. Houston, Downtown) has established a second site: (www.uhd.edu/academic/colleges/sciences/ns/pdresearch/index.html) which is being used for outreach to growers in Texas, where the Pierce's disease of grape is a major concern. 3. Genetic Data. We have significantly enlarged the genetic information available in our public MLST database at pubmlst.org/xfastidiosa. The data will continue to be increased as our analyses of the various forms of Xylella are published. All sequence data referenced in publications are also available from Genbank. 4. Data Correction. We have identified and communicated to the research community serious problems concerning two important Xylella resources: first, that the culture collection ATCC was providing the wrong genotype (in fact the wrong subspecies) in place of a strain commonly used in experimental work (Temecula-1); and second, that the draft genome of the strain Ann1, previously used in several research publications, was actually a mix of two different subspecies. PARTICIPANTS: Leonard Nunney (PI). His primary goal has been the ongoing planning and oversight of the project, data analysis, and writing manuscripts. Stephanie Russell (SRA). Her role has been maintaining the culture collection, sample isolation, DNA sequencing, and database management. Hamid Azad (SRA). His role has been to run the plant-host specificity experiment, including the maintenance of plants, monitoring disease progression, and destructive sampling of plants to determine the spread of Xylella using ELISA. Samia Elfekih (Visiting post-doctoral researcher). During her short stay (from Tunisia), she learnt the techniques necessary for MLST (isolation of cultures, DNA sequencing), and contributed to the writing of our most recent paper on MLST. Jennie Sotelo (Student Helper/Lab Assistant). She began working for us as an undergraduate, and continued after graduation while she applied to PhD programs. She was involved in the isolation and typing of Xylella isolates and initiated a side project examining the nutritional requirements of different forms of Xylella. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
During this year we have continued to work on developing a rigorous framework for monitoring Xylella. This effort has focused on documenting genetic variation, providing an understanding of the origins of this variation, and how it is linked to the infection of different plant hosts. We have now completed multilocus sequence typing (MLST) on almost 400 isolates from the US, Brazil, and Costa Rica. We have recently written a paper detailing the importance of MLST in microbiological studies such as this (Nunney et al. 2012). Last year we concluded that subspp. fastidiosa and sandyi were introduced into the US. In particular, subsp. fastidiosa, that causes an economically damaging disease of grape (Pierce's disease, PD), had been assumed to be native; however, our data are consistent with a single genotype being introduced from Central America around 150 years ago (Nunney et al 2010; Yuan et al 2010). This year (2011) our focus has been on the other two subspecies, subsp. multiplex, that we conclude is native to the US, and subsp. pauca, that is restricted to S. America. We have typed 112 isolates of subsp. multiplex and shown that different MLST sequence types (STs) show strong plant-host associations (Nunney et al, draft MS). We completed our analysis of subsp. pauca after typing 78 isolates from Brazil. This subspecies causes citrus variegated chlorosis (CVC), a serious disease of citrus, and is considered a major biosecurity threat to the US (it is listed as a USDA select agent). Our most important conclusion is that the form of subsp. pauca causing CVC is in fact an inter-subspecific recombinant between subspp. pauca and multiplex. We proposed that this mixing was made possible by the relatively recent introduction of subsp. multiplex into South America, probably in the 1970s (Nunney et al, Submitted). This example highlights our thesis that inter-subspecific recombination is an important mechanism leading to the infection of novel hosts. This view is further supported by our analysis of mulberry-type isolates in the US. Our MLST of 17 mulberry types from across the US (DC to CA) showed them to be (a) genetically homogeneous but (b) a recombinant mix of two subspecies, fastidiosa and multiplex. To confirm this view we sequenced the complete genome of one mulberry type. Analysis is not yet complete but our initial estimate is that it took at least 200 homologous recombination events to create this genome. No other forms of Xylella have been isolated from mulberry. Our experimental study of the plant-host specificity of the different subspecies and MLST sequence types within subspecies has been ongoing for 5 years at two locations (Apopka, FL and Riverside, CA) across a standard set of plant hosts. Unlike previous studies, we can test for variation in infection due to geographical location and year-to-year differences. Our general conclusions are that some plant hosts may be driving specialization (e.g. grape, oleander, mulberry, peach, citrus), while others may permit mixing of the different genotypes (almond, plum). The first of a series of manuscripts detailing this work is currently being written.
Publications
- Nunney, L., S. Elfekih and R. Stouthamer. 2012. The importance of multilocus sequence typing: cautionary tales from the bacterium Xylella fastidiosa. Phytopath. (In Press).
- Nunney,, L., X. Yuan, R. E. Bromley, and R. Stouthamer 2012. Inter-subspecific recombination and host specificity of Xylella fastidiosa in Brazil. Appl. Exp. Microbiol. (Re-submitted following revision).
|
Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: 1. Conference Presentations. PI L. Nunney gave the annual Plant Biosecurity presentation at the 2009 American Phytopathological Society meeting (July 2009, Portland, Or). At that conference, he also presented a second talk entitled "Central American origin of the bacterial pathogen causing Pierce's disease". In June 2009, he presented a talk at the national meeting of the Society for the Study of Evolution entitled "Homologous recombination and the invasion of a new plant host by the pathogenic bacterium Xylella fastidiosa". In December 2009 at the annual Pierce's Disease meeting in Davis, Ca, he (together with co-PI R. Stouthamer and lab staff, R. Bromley and S. Russell) presented a poster "An interactive website dedicated to the study of Xylella fastidiosa: www.xylella.org)" and a second poster (together with co-PI R. Stouthamer and Collaborator L. Morano, post-doc X. Yuan, and graduate student S. Spring-Pearson) "MLST analysis of Xylella fastidiosa isolates causing Pierce's disease of grape across the United States". 2. Websites. We have two websites to provide information about Xylella and the plant diseases that it causes, to introduce the goals of the project, and to solicit samples from plants exhibiting suspected Xylella infection. The first site (www.xylella.org) is our primary website for agricultural professionals to report leaf-scorch disease outbreaks, to which we will respond with sampling kits. This year we added an interactive map so that users can identify locations of isolates previously found in specific plant hosts. Collaborator L. Morano has established a second site: (www.uhd.edu/academic/colleges/sciences/ns/pdresearch/index.html) which is being used for outreach to growers in Texas, where the Pierce's disease of grape is a major concern. 3. We have significantly enlarged the genetic information available in our public MLST database at pubmlst.org. The data will continue to be increased as our analyses of the various forms of Xylella are completed. 4. We have established an important collaboration with researchers at the Univ. Costa Rica. PI L. Nunney and H. Azad (lab staff) visited the lab (headed by L. Moreira) to collect samples and discuss potential joint work on Xylella. 5. We have established a collaboration with Los Alamos National Labs (LANL) to sequence additional Xylella genomes. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
To develop effective Xylella monitoring we require an efficient detection system and a rigorous framework for interpreting the data. To these ends, we are currently focusing on the following questions: 1. What are the biogeographic patterns of Xylella We have completed multi-locus sequence typing (MLST) on more than 350 isolates from a broad geographical range across the southern US, from Brazil, and from Costa Rica. We are confident that two of the subspecies (fastidiosa, causing Pierce's disease, and sandyi, causing oleander leaf scorch) were introduced as single genotypes into the US a short time before 1880 and 1980 respectively. This is contrary to the prevailing view that they are native to the US. In support of our conclusion, we have completed a whole genome comparison between the two sequenced genomes of subsp. fastidiosa (Temecula-1 and M23), demonstrating that they evolved from a common ancestor in the very recent evolutionary past - consistent with our introduction hypothesis. Our MLST of 24 isolates from Costa Rica demonstrated that subsp. fastidiosa is genetically diverse in Central America, indicating that it is native to Central America, where it has been isolated primarily from coffee and grapevine. Circumstantial evidence indicates that subsp. fastidiosa was introduced into Southern California in an infected coffee plant. Even though subsp. fastidiosa is now widespread in the US, further introductions should be avoided, since they would introduce novel genetic variation that could undermine the measures being taken to control Pierce's disease (e.g. the development of resistant grapevines). 2. How host specific are the forms of Xylella We have developed a protocol for documenting host range, using a standard set of host plants (grapevine, oleander, almond mulberry, oak, elm, crapemyrtle, peach, plum and sycamore). We have completed two years of testing at two locations (Apopka, Florida and Riverside, California) and have initiated a third year. The preliminary conclusions are that some plant hosts may be driving specialization (e.g. oleander, mulberry), while others may permit mixing of the different genotypes (e.g. almond, plum). This work is undermines the long-held belief that Xylella is a host generalist. Although this bacterium infects a very wide range of plants, in some cases at least, only specific genotypes can infect specific hosts. 3. What is the mechanistic and ecological basis of inter-subspecific recombination (ISR) We have evidence that ISR is restoring genetic variation in subsp. fastidiosa, and that some isolates of subsp. multiplex carry genetic sequence from subsp. fastidiosa. However, the strongest evidence for the importance of ISR comes from our MLST of mulberry-type isolates. This form resulted from massive recombination between subspp. fastidiosa and multiplex. We are collaborating with LANL to sequence the mulberry-type genome to detail this unexpected genetic mixing. We have shown that this is the only form of Xylella that infects mulberry, so this finding strongly supports our hypothesis that ISR is important because it can lead to plant-host switching and hence new forms of disease.
Publications
- No publications reported this period
|
Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: 1. Conference Presentations. PI Nunney gave the annual presentation at the 2008 American Phytopathological Society meeting (July 28, 2008, Minneapolis, Mn). He also presented a talk entitled: "Understanding Xylella plant-host specificity via evolutionary genomics" at a joint UC Riverside/Los Alamos National Laboratory (LANL) conference (Sept. 3, 2008) that has resulted in a collaborative genome sequencing project (see below). We presented a poster at the 2008 Pierce's Disease Research Symposium (Dec 15-17 2008, San Diego, Ca) entitled: "Phylogenetic analysis of Costa Rican strains of Xylella fastidiosa using MLST". The poster presented our important finding that X. fastidiosa isolates from Costa Rica are from the same sub-specific group that causes Pierce's disease (PD) in the US supporting our hypothesis that PD is an introduced disease. 2. Websites. We have two websites to provide information about Xylella and the plant diseases that it causes, to introduce the goals of the project, and to solicit samples from plants exhibiting suspected Xylella infection. The first site (www.xylella.org) will be our primary website for agricultural professionals to report leaf-scorch disease outbreaks, to which we will respond with sampling kits. At present, some informational pages are in place, but the data collection part of the site has not been activated since we are still refining the sampling kit. We have also established a second site (www.uhd.edu/academic/colleges/sciences/naturalscience/pdresearch/PD_ Home_Page.html) which is being used for outreach to growers in Texas, where the Pierce's disease of grape is a major concern. 3. We have established an MLST database at pubmlst.org. The data will become incrementally available to the public as our analyses of the various Xylella groups are accepted for publication (see below). PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have focused on the following questions to effectively monitor Xylella: 1. How much Xylella variation exists within the US and overseas We have now completed multi-locus sequence typing (MLST) on more than 200 isolates from a broad geographical range (CA, DC, FL, GA, KY, NC, and TX in the US, Brazil, Costa Rica and Mexico). This dataset is now adequate for meaningful analyses of the genetic patterns seen in Xylella, and we are preparing 5 manuscripts based on these data, 3 of which are in final draft form. Our most important conclusions are: that two of the subspecies (fastidiosa and sandyi) were introduced into the US, contrary to the prevailing view that they are native; and that recombination among subspecies can result in the infection of novel plant hosts. We are collaborating with researchers at the Univ. of Costa Rica to study the important reservoir of Xylella variation in Costa Rica. 2. How host specific are the forms of Xylella We are developing a protocol for documenting host range, using a standard set of host plants. We have completed 2 years of testing at 2 locations (Apopka, FL and Riverside, CA). One important goal is to find hosts that are diagnostic for specific genotypes. Current results indicate that diagnostic infections are: grape for PD type (subsp. fastidiosa), oleander for oleander type (subsp. sandyi), mulberry for mulberry type (a recombinant form), and peach for peach type (one group within subsp. multiplex). Almond and plum are permissive hosts in which diverse forms of Xylella can grow. It appears that some plant hosts are driving specialization, while others may permit mixing of the different genotypes. A manuscript detailing this work is in revision. 3. Can we reliably detect Xylella DNA in infected leafhoppers and plants This has proved to be the most difficult aspect of the project. Since we need to avoid moving live bacteria around the country, we have been developing DNA extraction methods that can be used by non-experts and mailed to our lab. We are collaborating with researchers at the LANL to exploit the latest detection technologies. We have developed methods that specifically identify the South American subspecies (pauca), which is a serious potential threat to our citrus industry. However, the main problem remains consistent detection (i.e. close to 100%) from plants and leafhoppers that we know to be infected. 4. Does inter-subspecific recombination drive host shifts We have examined 3 subsp. fastidiosa genomes (Temecula-1, M23 and Hopland) for evidence of recombination with subsp. multiplex, using comparative genomic hybridization and direct sequence comparison. We found 5-10 recombinant regions per genome (each averaging 2.5kb in length) which is consistent with transformation. Thus if insect or plant hosts are co-infected with multiple forms of Xylella, genetic recombination is possible and may be common. Indeed our data from the mulberry type Xylella shows that adaptation to mulberry was associated with massive recombination. These results reinforce our need to monitor for novel recombinant forms that may become established in new plant hosts.
Publications
- No publications reported this period
|
Progress 01/01/07 to 12/31/07
Outputs
1. Conference Presentation. PI Nunney gave an invited presentation at the 2007 Pierce's Disease Research Symposium (Dec 12-14 2007, San Diego, Ca) entitled: "Monitoring XYLELLA FASTIDIOSA for changes in host range and virulence using genome-based methods". A pdf copy of this talk will be available for public download from the CDFA (California Department of Food and Agriculture). The talk outlined the goals and initial results from the study and encouraged those interested in Xylella and Xylella-caused diseases to participate in our monitoring program. 2. Websites. We have developed two websites to provide information about Xylella and the plant diseases that it causes, to introduce the goals of the project, and to solicit samples from plants exhibiting suspected Xylella infection. The first site (www.xylella.org) will be our primary website for agricultural professionals to report leaf-scorch disease outbreaks, to which we will respond with sampling kits. At present,
the home page is in place, but the data collection part of the site has not been activated since we are still refining the sampling kit. We have established a second site (www.uhd.edu /pdresearch) which is being used for outreach to growers in Texas, where the Pierce's disease of grape is a major concern.
Impacts
An effective Xylella monitoring project requires an efficient detection system and a rigorous framework for interpreting the data. To these ends, we have focused on the following questions: 1. How much Xylella variation exists within the US? We have completed MLST (multi-locus sequence typing) on 89 isolates (adding to 26 completed earlier) from CA, TX, KY, GA, and FL. Despite our focus on isolates from undersampled plant hosts, 73% were non-recombinant (33% subsp multiplex; 31% subsp fastidiosa; 9% subsp sandyi). The remainder showed evidence of inter-subspecific recombination. We found three recurring recombinant forms, one each on mulberry (MUL: 4 isolates), purple-leaf plum (PLP: 3 isolates), and sweetgum (3 isolates), supporting our hypothesis that inter-subspecific recombination can lead to plant-host switching and hence new forms of scorch disease. 2. How host specific are the forms of Xylella? We are testing a standard protocol for documenting host range using
4 Xylella isolates (the 3 subspecies plus MUL). Each isolate was tested on 7 different host species (grape, almond, oleander, mulberry, crape myrtle, elm, and oak) in both CA (Riverside) and FL (Apopka). Initial results show the isolates of subspp. fastidiosa and sandyi successful only on their natural hosts of grape and oleander respectively. The almond isolate of subsp multiplex used was successful only on almond in CA, while it also showed some growth on grape in FL. The mulberry isolate was successful only on mulberry in CA, but has been most successful on almond in FL. No isolates have become established on crape myrtle, elm, or oak - all species known to support Xylella. Thus the initial results are consistent with the view that, although Xylella is renowned for its broad plant host range, specific isolates are narrowly specialized. 3. Can we reliably detect Xylella DNA in infected leafhoppers and plants? Since we need to avoid moving live bacteria around the country, we have
been developing DNA extraction methods that can be used by non-experts and mailed to our lab. We are collaborating with researchers at the Los Alamos National Laboratory to exploit the latest detection technologies involving antibody trapping that are effective at very low bacterial counts (a few tens of bacteria). We are also developing methods that specifically identify the South American subspecies (pauca), which is a serious threat to our citrus industry. Lab tested rtPCR primers are currently undergoing field testing in Brazil. 4. What is the mechanistic and ecological basis of inter-subspecific recombination? Using array-based comparative genome sequencing, initial analysis of two isolates (MUL and PLP) indicates that the average size of recombinant fragments (>600bp) is about 850bp. Such short fragments indicate that the likely source of genetic exchange is transformation, resulting from Xylella importing fragments of DNA from the environment. Thus if insects or plant hosts are
co-infected with multiple forms of Xylella, genetic recombination is possible and may be common. This conclusion reinforces our need to monitor for novel recombinant forms that may become established in new plant hosts.
Publications
- No publications reported this period
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