Progress 10/16/15 to 09/30/20
Outputs
Target Audience:The target audience for this work includes plant breeders, plant pathologists (especially those that work on common bean) and those involved in common bean production Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This work was performed by a Project Scientist and a Laboratory Assistant, with the Project Scientist charged with setting up and conducting the experiments. This provided professional development in terms of strengthening the research and mentoring experience. The Laboratory Assistant gained experience in new techniques. How have the results been disseminated to communities of interest?Professional societies and academics through journals published What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
A comprehensive screening of a differential set of common bean germ plasm with resistance to bean golden yellow mosaic virus (BGYMV) was performed with three geminiviruses (two begomoviruses and a curtovirus) via agroinoculation. Here, we used our existing agroclones of bean golden mosaic virus (BGYMV-MX), bean dwarf mosaic virus (BDMV) and beet curly top virus (CFH) to screen these differentails (91-9, A 429, Cardinal, DOR 309, Etna, G 122, L 10-2, Sanilac, T 39, TC-75, Yolano and Othello) that were kindly provided by Dr. Shree Singh. Note that the majority of these materials are of Middle American origin. In terms of the agroinoculation method, we used the needle-puncture method in which the shoot apex region of been seedlings at the stage where the first trifoliolate leaf is half-expanded. In our experience, this method provides the highest efficiency of infection, though we have also found that Andean materials tend to be more efficiently infected via agroinoculation that Middle American ones. Plants agroinoculated with BCTV were maintained in a greenhouse, whereas those inoculated with BDMV and BGMV were maintained in a growth chamber. Plants were evaluated on a 0-4 scale (0=no symptoms, 1=very mild, 2=moderate, 3=severe and 4=very severe) 30 days post inoculation (dpi). Three independent experiments with 20 plants per experiment (60 total) were used with BCTV, whereas 2 experiments with 10 plants each (20 total) were used for BDMV and BGYMV. Overall mean disease ratings were calculated, with ratings <1 considered resistant, 1-2 moderately resistant and >2 susceptible. For BCTV, only one material (DOR 390) was resistant (rating of 1.0), whereas A 429, T 39, TC-75, and Othello were moderately resistant. For BDMV, A 429 (0.3), Sanilac (0.5, and DOR 390 (1.0) were resistant, whereas 91-1, Cardinal, Etna, G 122, T 39, TC-75, Yolano and Othello were moderately resistant. Only a single material, L-10-2, was susceptible and that had a rating of 2.25. These results are consistent with high levels of resistance to BDMV in Middle American germ plasm. For BGYMV, all materials were resistant (A-429 [0.7], Cardinal [0.6], DOR 390 [0.4], Sanilac [0.7], T 39 [1.0, TC-75 [0.7], and Othello [0.2]) or moderately resistant (91-1, Etna, G 122, L-10-2, and Yolano). The results for BGYMV revealed considerable levels of resistance, and are fully consistent with these materials representing a series of differentials with resistance to BGYMV. Interestingly, nearly all of these materials also showed resistance to BDMV, whereas there were fewer materials with resistance to BCTV. DOR 390 was resistant to all three geminiviruses in this screening, whereas A 429, T 39, TC-75 and Othello were resistant/moderately resistant to all three viruses. These results indicate broad spectrum geminivirus resistance in some materials, which is in agreement with previous studies. Thus, there are numerous options for breeding opportunities and some of these materials have been used in these programs, e.g., DOR 390. It is also desirable to have these materials screened in trials where they are subject to inoculation with the insect vectors (i.e., beet leafhoppers for BCTV and whiteflies [Bemisia tabaci) for BGYMV and BDMV. We will analyze these results and work with breeders to identify commercial or close to commercial materials that we could then test against these three viruses to see if it has 'broad spectrum' geminivirus resistance in our nexThese materials also should be screened against BGMV.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Naito, F. Y. B., Melo, F. L., Fonseca, M. E. N., Santos, C. A., F., Chanes, C. R., Ribeiro, B. M., Gilbertson, R. L., Boiteux, L. S. and de Cassia Pereira-Carvalho, R. 2019. Nanopore sequencing of a novel bipartite New World begomovirus infecting cowpea. Arch. Virol. 164: 1907-1910.
|
Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The target audience for this work is researchers involved in common bean breeding and production and pathology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project was carried out by a post-doctoral research associate, Dr. Monica Macedo, who received training in methods of inoculation of common beans with cloned geminivirus DNA and other aspects of the molecular characterization of these viruses. This training helped Dr. Macedo obtain a faculty position in Brazil at Federal Institute of Education, Science and Technology Goiano, Campus Urutai, Goias, Brazil. How have the results been disseminated to communities of interest?These results have been reported as part of a presentation given by Dr. Macedo at the 9th International Geminivirus Symposium held at UC Davis from November 9-13, 2019. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue to assess the role of crops and weeds as inoculum sources for bean-infecting geminiviruses as part of a larger effort to develop IPM packages to manage these viruses.
Impacts
What was accomplished under these goals?
Whitefly-transmitted begomoviruses, especially Bean golden mosaic (BGMV) and Bean golden yellow mosaic virus (BGYMV). continue to be major constraints on common bean production in tropical and subtropical regions of the New World. This included counties such as Brazil, the Dominican Republic and the USA. We have established that an effective means of managing begomoviruses with narrow host ranges is the implementation of a mandatory host-free period. This has had some success in managing BGYMV in the Dominican Republic where common bean production occurs primarily in a single isolated valley (San Juan de la Maguana). This strategy is much more difficult to implement in Brazil, where there are adjacent and overlapping crops of common bean, soybean and tomato, all of which are hosts of the whitefly, Bemisia tabaci MEAM 1. What is less clear is whether the host ranges of the viruses overlap as well. Here, we tested the hypothesis that common bean and soybeans are not hosts of the predominant tomato-infecting begomovirus, Tomato severe rugose virus (ToSRV), based on the failure to observe symptoms in these leguminous hosts when planted near tomato fields with ToSRV-infected plants. If so, this would indicate that common bean and soybeans could be grown during the tomato-free period aimed at reducing inoculum sources of ToSRV. Surveys of common bean and soybean plants in fields near tomato fields in which some plants were infected with ToSRV were conducted and leaves were collected randomly from symptomless plants. For both common bean and soybean samples, ToSRV infection was detected in a small number (<5%) of plants based on PCR tests with ToSRV-specific primers. Sequence analysis of selected PCR-amplified fragments from common bean and soybean were >97% identical to sequences of ToSRV, consistent with the capacity of this virus to infect these leguminous hosts. Next, to confirm that ToSRV can infect common bean, bean plants (cv. Topcrop) were agroinoculated with the infectious cloned DNA-A and DNA-B components of an isolate of ToSRV. Controls were bean plants (cv. Topcrop) agroinoculated with the infectious cloned DNA-A and DNA-B components of BGMV-BR-CAM, tomato plants (cv. Glamour) agroinoculated with ToSRV, and bean and tomato plants agroinoculated with A. tumefaciens with the empty vector. Twenty-one days after agroinoculation with ToSRV, common bean plants did not show obvious symptoms, but all plants were infected based on detection of ToSRV by PCR with the specific primer pair. Tomatoes agroinoculated with ToSRV developed typical symptoms of infection (mosaic and mottling), common bean plants agroinoculated with BGMV developed typical golden mosaic symptoms and plants agroinoculated with the empty vector did not develop symptoms nor was geminiviral DNA detected in these plants by PCR with ToSRV-specific primers. Taken together, these results show that common bean is a symptomless host of ToSRV and suggest that such plants could serve as a source of inoculum for ToSRV during the tomato-free period. In the case of soybeans, which are recalcitrant to agroinoculation, particle bombardment was used to inoculate soybean seedlings with ToSRV. Here, ToSRV was also found to induce a symptomless infection in soybean, similar to the situation in common bean. Tomato plants bombarded with the cloned infectious DNA components of ToSRV developed typical symptoms of mosaic and mottle, whereas common bean and soybean plants bombarded with gold particles only developed no symptoms. Furthermore, to test if soybeans infected with ToSRV could serve as a source of the virus for whiteflies, transmission experiments were conducted. Here, whiteflies (~50 adults from a 'clean' colony) were given a 48 hr acquisition access period on ToSRV-infected (symptomless) soybeans infected with ToSRV by particle bombardment and these whiteflies were provided a 48 hr inoculation access period on soybean or tomato plants. By 21 days post-exposure to whiteflies, soybean plants did not show symptoms, but 60% were positive for ToSRV based on PCR with the specific primer pair. For tomato plants exposed to equivalent whiteflies, ~70% developed typical mosaic and mottle symptoms by 21 after exposure. Thus, these results showed that not only did ToSRV induce a symptomless infection in soybeans, these plants could serve as inoculum sources of ToSRV for whiteflies to acquire the virus and transmit it to tomato plants. Together, these finding reveal that leguminous crop plants may serve as inoculum sources for a tomato begomovirus during the tomato-free period in Brazil. These findings reveal the importance of carefully assessing the host range of a begomovirus before selecting the plants to be included in a host-free period.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Macedo, M., Barreto, S. S., Costa, T. M., Maliano, M. R., Rojas, M. R., Gilbertson, R. L., and Inoue Nagata, A. K. 2017. First report of common beans as a non-symptomatic host of Tomato severe rugose virus in Brazil. Plant Dis. 101: 261.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Macedo, M. A., Barreto, S. S., Costa, T. M., Rocha, G. A., Dianese, E. C., Gilbertson, R. L., and Inoue-Nagata, A. K. 2017. First report of Tomato severe rugose virus, a tomato-infecting begomovirus, in soybean plants in Brazil. Plant Dis. 101: 1959.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Rojas, M.R., Macedo, M.A., Maliano, M.R., Soto-Aguilar, M., Souza, J. O. et al. 2018. World management of geminiviruses. Ann. Rev. Phytopathol. 56: 637-76.
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The target audience for this work is researchers involved in common bean breeding and production and pathology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project was carried out by a Ph.D. student, Mike O'Leary, who graduated with his degree in March 2018. During his Ph.D., Dr. O'Leary developed into a competent bacteriologist as well as becoming well-trained in the field of genomics. This training should put him in an ideal position to continue his career. How have the results been disseminated to communities of interest?These results have been presented in Department seminar presented by Mike O'Leary. What do you plan to do during the next reporting period to accomplish the goals?We hope to write a manuscript on this work and submit it for publication.
Impacts
What was accomplished under these goals?
Strains of Pseudomonas syringae pv. phaseolicola (Psp) have been described that produce a brown diffusible pigment in culture, particularly in strains originating from various African bean growing regions. Brown pigment produced by Psp strains has not been characterized biochemically, but it appears to be similar in appearance to pyomelanin produced by strains of Xanthomonas fuscans subsp. fuscans (Xff), the causal agent of common bacterial blight of bean. Pyomelanin is produced in Xff strains as the result of a loss-of-function mutation in the gene encoding homogentisate dioxygenase (hmgA), leading to the accumulation and oxidation of homogentisic acid to pyomelanin. Additionally, pyomelanin production can be conferred by a loss-of-function mutation of the hmgA gene in X. axonopodis pv. phaseoli strains, which normally do not produce pyomelanin. Thus, we hypothesized that pigmented Psp strains may also have a loss-of-function mutation in the hmgA gene, resulting in the accumulation and oxidation of homogentisic to pyomelanin. To test this hypothesis, we used the genome sequence of Psp 1448A (Bioproject ID: PRJNA12416; Accession CP000058 AAEZ01000000 AAEZ01000001-AAEZ01000028 AY922993 Version CP000058.1) to develop PCR primers to direct the amplification of an ~1.5 kilobase (kb) DNA fragment including the hmgA gene. This 1.5 kb fragment was PCR-amplified from the three pigmented Psp strains and a non-pigmented Psp strain originating from Wisconsin (strain HB114), and the sequence of the amplified ~1.5 kb fragment was determined for each strain. Sequence comparison were then made with the complete genome sequence of Psp1448A. These comparisons revealed that the non-pigmented HB114 strain and Psp 1448A sequence have a full-length and in-frame hmgA gene, whereas the three pigmented strains each had a single nucleotide (nt) deletion at nt 467 (out of 1308 total nts). This mutation results in a frameshift mutation that pseudogenized hmgA such that only the first 155 amino acids (aas) of the HMGA protein is translated (out of 435 aa residues in the wild-type protein), and 12 additional aas translated out-of-frame before a premature stop codon. Thus, as in Xff strains, brown pigment production in Psp strains is the result of accumulation and oxidation of HMGA to pyomelanin, due to a loss-of-function of the hmgA gene. The benefit, if any of brown pigment production by Xff and Psp remains to be determined.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
Bacterial blights of common bean: Application of traditional, molecular and genomic sequences for improved ID and understanding of mechanisms of pathogenicity
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The target audience for this work is researchers involved in common bean breeding and production and pathology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has been carried out by a Ph.D. student, Mike O'Leary, and this has provided him considerable experience in detection and characterization of plant pathogenic bacteria. This is helping him develop into a plant pathologist and bacteriologist. How have the results been disseminated to communities of interest?These results have been presented in Department seminar presented by Mike O'Leary. What do you plan to do during the next reporting period to accomplish the goals?We hope to write a manuscript on this work and submit it for publication.
Impacts
What was accomplished under these goals?
Halo blight (HB), caused by Pseudomonas syrinage pv. phaseolicola (Psp), and bacterial brown spot (BBS), caused by Pseudomonas syrinage pv. syringae (Pss), are foliar diseases of common bean (Phaseolus vulgaris) which co-occur in bean growing regions around the world. Psp and Pss are morphologically similar in culture, and thus must be differentiated through pathogenicity or molecular tests. To differentiate Psp, Pss, and non-pathogenic pseudomonads strains recovered from diseased bean tissue, we have developed a workflow that rapidly and reliably differentiates these strains. This workflow incorporates a tobacco hypersensitive response (HR) test and multiplex PCR, utilizing primers targeting toxin genes commonly synthesized by Psp and Pss strains: HB14f/r, which target a gene required for phaseolotoxin production by Psp strains, and B1/2, which target a gene required for syringomycin production by Pss strains. Amplification of the 1.4 kb HB14 band from a strain suggests that strain is Psp, while amplification of the 752 bp B1/2 band in conjunction with induction of HR on tobacco (cv. Havana) suggests that strain is a BBS-causing Pss strain. We applied this workflow to characterize pseudomonad strains recovered from a bean seed lot grown in Turkey in 2012, and a disease survey performed in bean growing regions of Turkey from 2013 through 2015. Pseudomonad strains were isolated on semi-selective mediums, sub-cultured, and preserved for further analysis. Three pseudomonad strains produced a diffusible brown pigment in culture, and were recovered from the bean seed lot grown in an unspecified region of Turkey in 2012. Forty-six pseudomonad strains were isolated from 30 leaf samples with symptoms of HB and/or BBS, each from an individual plant. Thus, a total of 49 strains were examined. All 49 strains were screened with the HB14 and B1/2 multiplex PCR. The 752 bp B1/2 band, but not the 1.4 kb HB14 band, was amplified from 23 strains recovered in 2015. These 23 strains also induced HR on tobacco (cv. Havana). This suggests that these strains are BBS-causing Pss, which was confirmed by the induction of BBS symptoms on leaves of common bean (cv. Topcrop) by these strains. On the other hand, the 1.4 kb HB14 fragment, but not the 752 bp B1/2 fragment, was amplified from the three pyomelanin-producing strains recovered from bean seed, and from 25 strains recovered from 2013-2015, indicating these are HB strains. The Psp strains collected in 2012 and 2013, including the three pyomelanin-producing strains, induced HR on tobacco (cv. Havana), and induced typical Psp symptoms on bean (cv. Topcrop). Tobacco HR response and pathogenicity were not determined for the Psp strains collected in 2014 and 2015. Finally, one strain isolated in 2015 generated bands with neither primer set, did not induce HR on tobacco (cv. Havana), and induced no symptoms on bean (cv. Topcrop). This is likely to be a non-pathogenic fluorescent pseudomonad. Thus, our workflow system for identification and differentiation of Pss and Psp successfully allowed for the identification of these bacteria from samples collected in Turkey, and revealed that both HB and BBS are present. This has important implications in management of bean blight bacteria in Turkey.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2017
Citation:
Gilbertson, R. L., O'Leary, M., Agarkova, I. V., and Vidaver, A. K. 2017. Detection of Xanthomonas campestris pv. phaseoli and X. fuscans subsp. fuscans in common bean seeds. Pages 63-71 in: Detection of Plant-Pathogenic Bacteria in Seed and Other Planting Material. Eds. M. Fatmi, R. R. Walcott and N.W. Schaad. APS Press. St. Paul, MN.
|
Progress 10/16/15 to 09/30/16
Outputs
Target Audience:The target audience for this work is researchers involved in common bean breeding and production and pathology.. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has been carried out by a Ph.D. student, Mike O'Leary, and this has provided him considerable experience in genomics, DNA sequencing and sequence analysis. This is helping him become proficient in these techniques, which will help him in his career. How have the results been disseminated to communities of interest?These results are preliminary and have not be disemminated yet. What do you plan to do during the next reporting period to accomplish the goals?We plan to complete the sequence analysis of Cff and continue of studies of the genetic diversity of bean pathogens and improved methods for diagnosis and disease resistance screening.
Impacts
What was accomplished under these goals?
Bacterial wilt is a seed-transmissible disease of common bean caused by the gram positive bacterium Curtobacterium flaccumfasciens pv. flaccumfasciens (Cff), and is found in bean growing regions across the globe. Symptoms include wilting and leaf scorch, which may superficially resemble symptoms caused by common bacterial blight (caused by Xanthomonas axonopodis pv. phaseoli and X. fuscans subsp. fuscans). Seed produced by infected plants may be shriveled or discolored. Bacterial wilt caused significant damage to bean production in the Midwest up until the 1970's, when it ceased to be a serious issue. However, in the early 2000's bacterial wilt reemerged as a problem impacting production in bean growing regions in Colorado, Nebraska, and Wyoming. During a disease survey conducted in bean fields in Turkey in 2015, leaf samples with symptoms resembling CBB were collected and applied to FTA cards. From some samples, no CBB bacteria were recovered on media semi-selective for CBB (i.e., MXP); however, yellow bacteria were recovered on general growth media (i.e., 523). These bacteria were determined to be gram-positive based on KOH reaction. Ribosomal internal transcribed spacer fragment sequence analysis indicated these bacteria were likely Cff. Despite being recovered from the same field the five Cff isolates were not genetically uniform, with each isolate generating a unique DNA fingerprint with BOX primers. This is consistent with a published analysis of diversity among Cff isolates collected over 50 years across Colorado, Iowa, and Nebraska, which showed significant genetic variability among isolates via DNA fingerprinting, but found no correlation with phenotypic appearance (e.g., color), geographic origin, or year of isolation. Genome sequences are a powerful research tool for working with plant pathogenic bacteria. When this project was initiated, no genome sequence was publicly available for Cff, and only one draft genome was available for any Curtobacterium species. This strain, C. flaccumfasciens UCD-AKU, was isolated from a residential carpet, not in association with plant material, and its status as a plant pathogen is unknown. Currently, several (25) draft genomes and two complete Curtobacterium genomes are publically available, but Cff lacks a published genome. To provide a resource to help understand the biology of Cff as a plant pathogen, we sequenced one isolate, Cff1037, via the Pacific Biosciences RSII platform using a single SMRT cell. The de novo assembly yielded two contigs (~3.7 Mb and ~115 kb, with 173x and 229x coverage, respectively), with an N50 read length of ~15 kb, average coverage of ~175x, and 92.8% of reads mapping to the assembly. The current draft assembly has a total size of ~3.8 Mb, and G+C content of 70.9%, both of which are typical of Curtobacterium species. Neither contig is obviously circular, based on dot plot comparisons of the 5' nor 3' ends of each contig. Current work is focused on finishing the assembly by determining if these contigs are part of the same molecule, and how they are oriented relative to each other. As with other gram positive bacterial pathogens, Cff1037 does not contain a type III secretion system. Interestingly, the genome of Cff1037 is predicted to contain at least one internalin-like protein. These proteins are known to be involved in pathogenicity of other gram positive bacteria, such as Listeria monocytogenes. However, the putative internalin found in Cff1037 has limited homology to internalins with known virulence functions, so its importance is unclear.
Publications
|
|