Progress 11/01/15 to 10/31/19
Outputs
Target Audience:The target audience for this effort includes agricultural and life scientists (plant pathologists) at universities, federal agencies and in the private sector (specifically vegetable seed companies). Additionally, the target audience includes graduate students and postdoctoral associates working in thearea of plant pathology and cooperative extension agents crop management advisors. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this project we trained 3 doctoral students and 4 international visting scientists. How have the results been disseminated to communities of interest?Research findings have been disseminated in peer-reviewed publications, and oral and poster presentatiosn at state, regional, national and internatonal conferences and symposia. Presentationshave also been made at invited departmental seminars in and outside the United States. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Goal 1. Using a non-pathogenic strain ofA. citrulli, we determined the optimal concentration of BCA cells needed to inoculate watermelon flowers to maximize seed treatment was 108CFU/flower. Through in vivo and in vitro screens, we identified two new BCA candidates (BCA#24 and BCA#35) with antagonistic activity againstA. citrulli. Through comprehensive molecular and physiological analyses, these strains were identified asBacillus safensis. Additionally, we acquired a known BCA,Bacillus mojavensisstrain RRC101. To determine the mode of action of theB. safensisandB. mojavensisstrains againstA. citrulli, we investigated if the strains produced antimicrobial compounds, displayed a competitive advantage, or induced systemic acquired resistance (ISR), which are commonly associated with biological control activity ofBacillusspp.B. mojavensisRRC101 produced a distinct zone of inhibition (ZOI) withA. citrulli, which suggested that it produces active compounds. In contrast, theB. safensisstrain did not produce a ZOI. To determine if competition was a component of biocontrol activity, we inoculated germinating watermelon seeds with the candidate BCAs individually and in combination withA. citrulli. When BCAs were co-inoculated withA. citrulli, theA. citrullipopulation growth was reduced by 2-3 orders two days after planting. However, when each strain was inoculated independently onto germinating seeds, theA. citrullipopulations increased to significantly higher populations than the BCAs. Finally, to determine if the BCAs induced systemic acquired resistance, we artificially inoculated melon seeds and measureLOX2gene expression at three and six days after planting. However, there was no consistent upregulation ofLOX2in melon seedlings from seeds inoculated with the BCAs. Finally, we investigated the efficacy of watermelon seed treatments with BCAs by flower inoculation under field conditions. Sixty-three watermelon seedlots primed with BCAs were generated by flower inoculation under field conditions and greater than 50% of them tested positive for BCA bacteria. Samples of each seedlot were challenged with 107CFU/ml ofA. citrullito determine if flower inoculation with BCA reduced BFB seed-to-seedling transmission. We observed a 35-40% reduction in BFB seedling transmission for seeds from flowers treated with BCA bacteria for two experiments conducted in Tifton GA. These treatments were significantly different from the water control treatments. However, in a third experiment conducted in Athens GA, there was no significant reduction in BFB incidence. Goals 2. During the early stages of watermelon seed germination,A. citrullicolonizes seed tissue in a saprophytic manner before seed-to-seedling transmission of BFB. To gain a better understanding of this process, we performed transcriptome analysis ofA. citrullicultured in watermelon seed homogenate representing different stages of seed germination. Watermelon seeds were germinated for 24, 48 and 120 h, then crushed to generate seed homogenates (SH). In 24 h SH, theA. citrullitranscriptome showed an overall response to nitrogen starvation. Because glutamine synthetase (GS) plays a central role in the bacterial nitrogen starvation response, we studied its role inA. citrullicolonization of germinating watermelon seeds. We observed that inA. citrullistrain AAC00-1, GS was up-regulated in response to nitrogen starvation and was expressed at a constant level in nitrogen rich growth media. AnA. citrullimutant (AAC00-1Δ3101) that had its uridylytransferase (Aave_3101) gene deleted was unable to up-regulate GS upon nitrogen starvation, but colonized germinating seeds at the wild type (WT) rate. On the other hand, the insertion GS mutant, AAC00-1GSin, displayed reduced seed colonization and reduced BFB seed-to-seedling transmission compared to the WT. These results suggested that basal GS expression, but not induced expression, is required forA. citrullicolonization of germinating watermelon seeds. Goal 3. We established field plots with four cucurbit species and introduced group I and IIA. citrullistrains as the inoculum sources. The results from the field trials confirmed thatA. citrullistrains exhibited a preference for different cucurbit species, which was more pronounced in fruit tissues. Group I and IIA. citrullistrains were inoculated on detached melon fruits by syringe- and swab-inoculation, and on attached melon fruits by swab-inoculation. From these artificial inoculations, we found that physical penetration, colonization, and symptom development did not account for the host preference ofA. citrullion melon fruits.Additionally, we did not find detectable effects of XopJ homologs onA. citrullivirulence on watermelon and melon cotyledons. The deletion mutant of XopJ homologs was less virulent on attached melon fruits compared to the wild-type, while their population levels on melon fruits were variable across experiments. However, the HopAF homolog may be important forA. citrullicolonization and symptom development on watermelon and melon cotyledons. Additionally, we developed an immature watermelon fruit assay to distinguish group I and IIA. citrullistrains and used it to identify virulence determinants ofA. citrulli. Only group II strains induced water-soaked lesions on immature watermelon fruit tissues. By screening anA. citrulliAAC00-1 transposon mutant library, we found six mutants were non-pathogenic on watermelon fruits. Three of these mutants were disrupted in type 3 secretion system associated genes. Goal 4. A citrullirequires a functional type III secretion system (T3SS) for pathogenicity and preliminary analysis of a sequencedA. citrullistrain revealed 11 Type II effector (T3E) genes. In partnership with Saul Burdman's team we sought to identify additional T3Esby genome sequence analysis of the A. citrulli strain M6. M6 contained 51 genes whose products are similar to known T3Es. The expression of these genes was compared using RNA Seq analysis and we identified seven novel T3E genes that encode hypothetical proteins that appear to be unique to plant pathogenicAcidovoraxspecies.?
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Traore, S.M., Eckshtain-Levi, N., Miao, J., Castro-Sparks, A., Wang , Z., Wang, K., Li, Q., Burdman, S., Walcott, R., Welbaum, G.E., Zhao, B. 2019. Nicotiana species as surrogate host for studying the pathogenicity of Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbits. Molecular Plant Pathology 20: 800-814 https://doi.org/10.1111/mpp.12792
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Yang, Y.W., Zhao, M., Zhang, L.Q., Qiao, P., Bai, X., Zhang, X.X., Walcott, R.R., Guan, W., and Zhao T.C. 2019. Development of a multiplex PCR assay based on the pilA gene sequences to detect different types of Acidovorax citrulli. Journal of Microbiological Methods 159: 93-98
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Liu, D., Cui, Y., Walcott, R.R., Diaz-Perez, J., Tishchenko, V., and Chen, J. 2019 Transmission of human enteric pathogens from artificially-inoculated flowers to vegetable sprouts/seedlings developed via contaminated seeds. Food Control. 99: 21-27
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Kan, Y., Jiang, N., Lyu, Q., Gopalakrishnan, V., Walcott, R., Burdman, S., Li, J.Q., and Luo, L. 2019. Induction and resuscitation of the viable but non-culturable (VBNC) state in Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbitaceous crops. Frontiers in Microbiology 10: 1081 https://doi.org/10.3389/fmicb.2019.01081
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Jiang, N., Lyu, Q., Han, S., Xu, X., Walcott R.R., Li, J.Q., and Luo, L. 2019. Evaluation of suitable reference genes for normalization of quantitative reverse transcription PCR analyses in Clavibacter michiganensis. Microbiology Open 8:e928 https://doi.org/10.1002/mbo3.928
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Tian, Y., Zhao, Y., Zhou, J., Sun, T., Luo X., Kurowski, C., Gong, W., Hu, B., and Walcott, R.R. 2019. Prevalence of Acidovorax citrulli in commercial cucurbit seedlots during 2010-2018 in China. Plant Disease 104: 255-259 https://doi.org/10.1094/PDIS-03-19-0666-RE
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Liu, J., Tian, Y., Zhao, Y., Zeng, R., Chen, B., Hu, B., and Walcott, R.R. 2019. Ferric Uptake Regulator (FurA) is required for Acidovorax citrulli virulence on watermelon. Phytopathology 109: 1997-2008 https://doi.org/10.1094/PHYTO-05-19-0172-R
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Yang, R., Santos-Garcia, D., P�rez-Monta�o, F., da Silva, G.M., Zhao, M., Jim�nez-Guerrero, I., Rosenberg, T., Chen, G., Plaschkes, I., Morin, S., Walcott, R. and Burdman, S. 2019 Complete assembly of the genome of an Acidovorax citrulli strain reveals a naturally occurring plasmid in this species. Front. Microbiol. 10, 1400. https://doi.org/10.3389/fmicb.2019.01400
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Jim�nez-Guerrero, I., P�rez?Monta�o, F., Da Silva, G.M., Wagner, N., Shkedy, D., Zhao, M., Pizarro, L., Bar, M. Walcott, R., Sessa, G., Pupko, T., and Burdman, S. 2020. Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III-secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the
Acidovorax genus. Molecular Plant Pathology 21: 1737 https://doi.org/10.1111/mpp.12877
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Zhao, M., and Walcott, R.R. 2020. Acidovorax citrulli is sensitive to elevated temperatures during early stages of watermelon seed germination. Seed Science and Technology 48: 11-20 https://doi.org/10.15258/sst.2020.48.1.02
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Zhao, M., Dutta, B., Luo, X., Burdman, S., and Walcott, R.R. 2020. Genetically distinct Acidovorax citrulli strains display cucurbit fruit preference under field conditions. Phytopathology 110: 973-980
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Zhang, X., Yang, Y., Zhao, M., Yang, L., Jiang, J., Walcott, R., Yang, S., and Zhao, T. 2020. Acidovorax citrulli Type III Effector AopP Suppresses Plant Immunity by Targeting the Watermelon Transcription Factor WRKY6. Frontiers in Plant Science, 11, 1723.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Liu D, Walcott R, Mis Solval K, Chen J. 2021. Influence of Bacterial Competitors on Salmonella enterica and Enterohemorrhagic Escherichia coli Growth in Microbiological Media and Attachment to Vegetable Seeds. Foods. 10(2):285.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Ge Y, Luo L, Xia L, et al. 2021. Fermentation: An Unreliable Seed Treatment for Bacterial Fruit Blotch of Watermelon. Plant Disease. 105:1026-1033.
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:University, Industry and Government plant pathologists. Seed Pathologists Graduate students Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Three PhD students are receiving extensive training and professional development in this project. Their PhD dissertation research projects are directly linked to various goals of this project. How have the results been disseminated to communities of interest?Results have been presented as posters or oral presentations at the American Phytopathological Society annual conference and the Georgia Association of Plant Pathologists annual meeting What do you plan to do during the next reporting period to accomplish the goals?We plan to repeat field studies to test the efficacy of flower inoculation with BCAs and investigate host specificity of group I and II A. citrulli strains on different ccucurbit species. We also plan to characterize specific T3S mutants to investigate the factors that contribute to host specificity in A. citrulli. We will continue to screen T3E mutants for lost of pathogenicity on watermelon fruits and gain of pathogenicity on melon fruits. Finally, we will determine the longevfity of BCAs in watermelon seeds amd determine if the flower inoculation BCA strategy is compatable with commercial seed production practices, including seed fermentation and antimicrobial seed treatments.
Impacts
What was accomplished under these goals?
Goal 1 To determine the mode of action for candidate biocontrol agents (BCAs), in vitro assays were conducted to screen for antagonistic activity against A. citrulli. BCAs were deposited onto 4 spots on agar plates that were previously inoculated with cell suspensions of A. citrulli. The non-pathogenic AAC00-1ΔhrcC strain was used as a negative control, and Serratia marcescens W11, a watermelon endophyte, was used as a positive control. Plates were incubated at 28°C for 48 h and the diameter of the zone of inhibition (ZOI) was measured for each spot. The average ZOI produced by the negative control and the two Bacillus safensis strains were ~0 mm. In contrast, the average ZOI produced by Serratia marcescens W11 was 48 mm and Bacillus mojavensis was 51mm. The mode of action for Bacillus mojavensis was antibiosis, likely due to a secondary metabolite production, while this was not the case for the B. safensis strains. BCAs were also evaluated for their ability to induce systemic resistance in melon seedlings. Cell suspensions ( ~ 108 CFU/ml) of BCAs were applied to the crown of four-week-old melon seedlings. Treatments included B. mojavensis RC101, B. safensis strain #35, AAC00-1ΔhrcC, and 50% Acibenzolar-S-methyl (247 ppm) as a positive control. Melon plants treated with water served as a negative control. At 0, 1, 2, 3, and 4 days after treatment, a single leaf was used for RNA extraction. RNA was used for cDNA synthesis and qPCR was utilized to analyze gene expression. ACT1 served as the endogenous control gene, and LOX2 (lipozygenase 2), served as a marker for ISR. Melon plants treated with water and Actigard displayed 2.0- and 1.38-fold increases in LOX2 expression by 3 days after treatment, respectively. Conversely, plants treated with B. mojavensis RC101, B. safensis strain #35, and AAC00-1ΔhrcC, displayed a 1.2-, 2.4-, and 0.7-fold increases in LOX2 expression, respectively. These BCAs trigger ISR and this may contribute to the mode of action against A. citrulli. This experiment is being repeated Goal 2 Based on A. citrulli RNASeq analysis, we hypothesized that the germinating watermelon seed is an ammonium nitrogen-limiting environment, and that the ability of A. citrulli to assimilate nitrogen is important for the colonization process. To investigate the role of the glutamine synthetase (GS) gene in A. citrulli colonization of germinating watermelon seeds we used homogenates of watermelon seeds that were germinated for 24 h, and 24 h watermelon seed exudates. Watermelon seed exudate was made from soaking watermelon seeds in water for 24 h followed by neutralizing the pH and filter-sterilization. Using qRT-PCR, AAC001 GS expression was confirmed to be up-regulated in watermelon seed homogenate and seed exudate compared to LB. GS expression was repressed by addition of NH4Cl or glutamine to the seed homogenate, confirming that the germinating seed was a nitrogen-poor microcosm. To further characterize the role of GS we attempted to make a GS knock-out A. citrulli mutant by inserting a plasmid in the GS gene. The resulting mutant was auxotrophic for glutamine; however, it was unstable in the absence of the selection pressure of the antibiotic kanamycin. Because of this, we were unable to characterize the colonization of the mutant strain on germinating watermelon seedlings in vivo. Instead, we are characterizing the population dynamics of this mutant in seed exudate amended with kanamycin. To identify additional genes that may be critical for A. citrulli colonization of germinating watermelon seeds, we screened mini Tn5-transposon insertion mutant library of AAC001. Mutants strains that were unable to colonize watermelon seed exudate were selected. Of 3000 strains screened, ~50 displayed delayed or no growth in watermelon seed exudate. The genes disrupted by Tn5 were identified, and Southern blot analysis was used to confirm single insertions. Based on the biological implication of the function of the disrupted gene, three strains were selected for further characterization. Goal 3 We sought to assess differences in cucurbit host preference between group I and II A. citrulli strains under natural field conditions. In 2018, two field plots, each with four cucurbit species (watermelon, melon, pumpkin and squash), were established in Tifton GA. By bacterial isolation and group-specific PCR assay, group I and II A. citrulli strains were recovered from leaf and fruit tissues expressing BFB symptoms. Forty two percent (28/67) of symptomatic watermelon leaf samples were infected with the group II strain, while 100% of symptomatic melon (n=80), pumpkin (n=2), and squash (n=19) leaf samples were infected with the group I strain. For fruit samples, no pumpkin or squash fruit lesions were determined to be positive for A. citrulli by PCR assay. On the other hand, 100% of the symptomatic melon fruit samples (n = 20) were determined to be infected with the group I strain. With regards to symptomatic watermelon fruits, 100% (2/2) were infected with the group I strain.Under natural field conditions, group II A. citrulli strains were only detected on watermelon foliage. In contrast, we detected group I A. citrulli strains in leaf lesions of all cucurbit hosts. We only detected group I A. citrulli strains in BFB symptomatic melon and watermelon fruits. The group II strain showed a preference for watermelon, while the group I strain showed little preference and infected all hosts. Statistical analysis of binary distribution data from 2017 and 2018 field studies using the GLIMMIX procedure showed that the odds of melon, pumpkin, and squash foliage being infected by the group I A. citrulli strain were 10.8, 7.6, and 11.7 times greater than the odds of watermelon foliage being infected by the group I strain, respectively. More strikingly, the odds of melon fruits being infected by the group I A. citrulli strain was 342 times greater than watermelon fruits being infected by the group I strain. To characterize the role of the putative type 3 secreted (T3S) effector XopJ in A. citrulli virulence and host preferential association single, double, and triple deletion mutants of Aave_2166, Aave_2708, and Aave_2938 were made by homologous recombination in the AAC00-1 background. Corresponding complements of the mutants were generated. In addition, transformants were made by conjugating Aave_2166 and Aave_2708 into the group I strain M6. Virulence tests of the XopJ triple mutant were conducted on melon and watermelon cotyledons and as expected, no significant difference was observed in bacterial population dynamics over time. The virulence of A. citrulli XopJ mutants on watermelon and melon fruits is currently being tested. Additionally, through comparative genome analysis of representative group I and II A. citrulli strains we identified 46 putative T3E genes based homology with known T3Es in other bacteria. To identify additional host preference and tissue specificity determinants unique to group II A. citrulli strains, we generated and screen a miniTn5 random insertion mutant library for strains that lost the ability to infect watermelon fruit. We generated 10,080 AAC00-1 transposon mutants and after screening 1,013 mutants, six strains were non-pathogenic on watermelon fruits. Additionally, these mutants did not cause disease on watermelon seedlings, nor induce HR on tobacco leaves. Two of the six strains were disrupted in hrpE and hrcN genes, that are part of the type III secretion apparatus. However, the other three were disrupted at a transposase gene, a type 6 secreted protein; the flagellar hook-associated protein 3; and a currently unidentified locus. Transposon mutants will be characterized in greater detail, followed by targeted gene deletion and characterization. Additionally, we will screen this library for mutant group II strains that have gained the ability to infect melon fruits.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Zhang, X., Zhao, M., Yan, J., Yang, L., Yang, Y., Guan, W., Walcott, R., and Zhao, T. 2018. Involvement of hrpX and hrpG in the virulence of Acidovorax citrulli strain Aac5, causal agent of bacterial fruit blotch of cucurbits. Frontiers in Microbiology 9: 507 doi: 10.3389/fmicb.2018.00507
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Giovanardi, D., Sutton, S.A., Stefani, E., and Walcott, R.R. 2018. Factors influencing the detection of Acidovorax citrulli in naturally contaminated cucurbitaceous seeds by PCR-based assays. Seed Sci. and Technol. 46: 93-106
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Liu, D., Cui, Y., Walcott, R., and Chen, J. 2018. Fate of Salmonella enterica and Enterohemorrhagic Eschericia coli cells artificially internalized into vegetable seeds during germination. Applied and Environ. Microbiol. 84: e01888-17
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The target audience for this work includes: 1) university researchers (plant pathologists and plant breeders) whose efforts will be informed by these findings 2) extension agents who can use this information todevelop effective management strategies for managing bacterial fruit blotch of cucurbit crops 3) seed pathoogists working for seed production companies; this information wil help them develop effective disease management strategies in seed production systems 4) vegetable farmers will benefit from having access to pathogen-free seeds and improved strategies for plant disease management. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Three PhD students are receiving extensive training and professional development in this project. Their PhD dissertation research is directly linked to various goals of this project. How have the results been disseminated to communities of interest?Results have been presented as posters or oral presentations at the American Phytopathological Society annual conference, the Georgia Association of Plant Pathologists annual meeting and the American Seed Trade Association annual meeting. What do you plan to do during the next reporting period to accomplish the goals?We will continue to evaluate flower application of BCAs under field conditions and evaluate the longevity of bacteria in stored seeds. We will also continue to investigate the role (if any) that induced systemic resistance in biocontrol by the Bacillus spp. We will generate glutamine synthetase mutants of A. citrulli and determine the role of nitrogen assimilation in watermelon seed colonization during the early stages of seedling colonization. Fianlly, we wil investigate the roles of specific type III secreted effector genes singly, or in concert, on the virulence of group II A. citrulli strains on melon fruit tissues. We will also screen a saturated transposon insertion library of AAC00-1 (group II) to identify genes that are critical for watermelon fruit infection.
Impacts
What was accomplished under these goals?
Goal 1. We determined that the optimal concentration for inoculating watermelon flowers to generate seed infected with the biocontrol agent (BCA) Acidovorax citrulli AAC00-1hrcC was 108CFU/flower. This inoculum load resulted in the highest percentage of seeds infested with the BCA and the highest concentraion of BCA per seed. Inoculating watermelon flowers with 108CFU/flower did not reduce the germination percentage of the resulting seeds. However, when challenged with pathogenic A. citrulli, BFB seed-to-seedling transmission was reduced to 20% relative to 45% for the negative control, whose flowers were inoculated with water. Screens to find naturally occurring BCAs in watermelon seed microflora revealed two species that were identified and Bacillus safensis by whole genome sequencing and Bacillus mojavensis. These strains were evaluated by watermelon flower inoculation under field conditions and seeds inoculated with these BCAs displayed significant reductions in BFB seedling transmission relative to the negative control flower treatment (phosphate buffered saline). Field studies will be repeated and efforts will be made to determine if BCA applied to flowers are compatible with antimicrobial seed treatments. We determined that the modes of action of B. mojavensis and B. safensis are in part antibiosis. However, we are still investigating if induced systemic resistance or systemic acquired resistance are factors in BFB biocontrol. Goal 2. We used RNASeq to compare the gene expression profiles of A. citrulli while colonizing watermelon seed exudate produced at 24, 48 and 120 h after imbibition. The transcriptome of A. citrulli in 24h seed exudate was significantly different to those generated in 48 and 120h seed exudate. 110 A. citrulli genes were consistently upregulated in 24 h watermelon seed exudate, relative to the other time points. More specifically, genes involved in nitrogen metabolism (e.g. glutamine synthetase) were significantly upregulated in 24 h watermlon seed exudate, suggesting that A. citrulli cells experience nitrogen starvation during the initial stages of seedling colonization. Using qPCR we showed that A. citrulli upregulates glutamine synthetase in low nitrogen conditions, which suports the hypothesis that glutamine synthetase expression corresponds to nitrogen stress. Research is in progress to use qPCR to measure glutamine synthetase expression during the early stages of in vivo A. citrulli colonization of watermelon seed and to generate A. citrulli glutamine synthetase knock-out mutants for characterization. Goal 3. We did not observe differences in virulence between group I and II A. ctrulli strains on seedlings of different cucurbit species in greenhouse assays. However, we observed that representative group I strains could cause lesions on immature detached fruits of melon cv Joaquin Gold, while group II strains could not. Similarly, group II strains induced watersoaked lesions on detached immature fruits of watermelon cvs. Crimson Sweet and Charleston Gray, while group I strains could not. These observations suggest that while there is no host specificty in foliar tissue (possible host preference), there are differences in host specificity between group I and II A. citrulli strains on fruit tissues. To test this hypotheis field studies were conducted in which melon, watermelon, pumpkin and squash plant were planted in a single plot and simultaneously exposed to group I and group II A. citrulli strain inoculum. Out of 9 watermelon fruits that developed BFB symptoms, 100% were infected with the group II strain. Of the 7 melons that developed BFB sysmptoms, 6 were infected with the group I strain, while 1 was infected with the group II strain. Additionally, using the detached melon fruit assay, we showed that group I strains require a functional type III secretions system for infection. This suggests that type III secreted effectors are determinants of host specificiaty in fruit tissues. We previously reported that group I and II A. citrulli strains differed in their arsenals of type III secreted effectors . We hypothesize that type III secreted effectors are important in determining the host range of A. citrulli groups. To test this hypotheis, we will expressing group II effectors from AAC00-1 in the group I A. citrulli strain M6 and evaluate if this alters virulence on melon and watermelon fruits. Results of these studies may reveal new targets for BFB resistance development.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Tian, Y., Zhao, Y., Chen, X., Dai, Y., Zhao, W., Hu, B., and Walcott, R.R. 2017. Evidence for a novel phylotype of Pseudomonas syringae causing bacterial leaf blight of cantaloupe in China. Plant Dis. 101: 1746-1752
- Type:
Books
Status:
Published
Year Published:
2017
Citation:
Detection of plant-pathogenic bacteria in seed and other planting material Second edition.2017. Fatmi, M., Walcott, R.R. and Schaad, N.W. APS Press
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Cui Y., Walcott, R. and Chen J. 2017 Differential attachment of Salmonella enterica and Enterohemorrhagic Escherichia coli to alfalfa, fenugreek, lettuce, and tomato seeds. Appl. and Environ. Microbiol. 83: e03170-16
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Yan, L., Hu, B., Chen, G., Zhao, M., and Walcott, R.R. 2017. Further evidence of cucurbit host specificity among Acidovorax citrulli groups based on a detached melon fruit pathogenicity assay. Phytopathology 101: 1305-1311
|
Progress 11/01/15 to 09/30/16
Outputs
Target Audience:The target audiences included research plant pathologists in academicand government institutions, and seed pathologist working in the vegetable seed industry. Our efforts were also directed toward commercial vegetable and transplant producers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Currently, three doctoral students are being trained on various aspects of this project. Additionally, we have hosted 3 visiting doctoralstudents (2 from China and 1 from Brazil) and one visiting scientist from Italy. How have the results been disseminated to communities of interest?Results have been dessiminated by four departmental seminars and three international symposia in S. Africa, South Korea and Mexico. What do you plan to do during the next reporting period to accomplish the goals?? Objective 1: Repeat field and greenhouse experiments to assess the efficacy of Bacillus spp. as seed treatments applied internally by flower inoculation against BFB, gummy stem blight and Rhizoctonia damping off. We will also characterize the mechanisms of biocontrol including antagonism and induced systemic resistance. Objective 2: Conduct qPCR analysis to confirm the upregulation of glutamine synthetase in A. citrulli cells during the first 24h of watermelon seed germination. Generate glutamine synthetase mutants of A. citrulli and determine their ability to colonize nitrogen rich and poor media, and germinating watermelon seeds. Objective 3: Generatedouble and triple T3E (XopJ) deletion mutants of the group II A. citrulli strain, AAC00-1 and testtheir pathogenicity and virulence in detached melon fruit assays. Generate and screen a random mini-Tn5 transposon insertion mutant library of AAC00-1 strains for the ability to infect detached immature melon cv Joaquin Gold fruits. This may identify genes that are involved in host range restriction in group II A. citrulli strains.
Impacts
What was accomplished under these goals?
Objective 1: Under field and greenhouse conditions, we demonstrated that inoculation of watermelon flowers with theBacillusspp (Bacillusspp. strains 24 and 35 andBacillus mojavensisRRC 101) cell suspensions (107CFU/flower) resulted in seed infestation with the biocontrol agents. Watermelon seed quality was not affected by infestation with these bacteria. When seed samples from these lots were challenged withA. citrulli(vacuum-infiltration in suspensions with 106CFU/ml) BFB seedling transmission was reduced by all strains relative to the negative control (seeds from female flowers treated with 0.1 M PBS buffer). In these trails the most effective biocontrol strain wasB.mojavensisRRC 101. Interestingly, the Bacillus spp. also demonstrated in vitro and in vivo antagonistic activity against fungal watermelon pathogens includingDidymella bryoniae(gummy stem blight) andRhizoctonia solani(Rhizoctonia damping off). These results suggest that after optimization, watermelon flower inoculation with biocontrol Bacillus spp., may contribute to an effective integrated pest management strategy for BFB and other seed and soilborne diseases of watermelon. Objective 2: Based on transcriptome analysis, we determined thatA. citrulligene expression activity was higher during the first 24 h of incubation in germinating watermelon seed exudate than after 48 and 120h. We observed upregulation of glutamine synthetase in A. citrulli cells growing in watermelon seed exudate, which suggests that during initial stages of germinationthe pathogen might be experiencing nitrogen limiting conditions. . Objective 3: We determined that the detached immature melon (cv Joaquin Gold) assay consistently distinguishes group I and II A. citrulli strains. More specifically, group I strains colonized and infected melon fruit tissues but group II strains did not. While this differential pathogenicity was observed on melon cv. Athena, it was not observed withother cucurbit speciesincluding melon cv. Magellan, cucumber cv. Long Green Improved and squash cv. Early Prolific Straightneck. Interestingly, the ability of group I A. citrulli strains to infect immature melon fruits was dependent on a type three secreted effectors (T3E), asa group I strain impaired in type 3 secretion (M6 delta hrcV) failed to infect immature melon fruit tissues. Based on these observations, we hypothesize that the difference in T3S effectors possessed by group I and II A. citrulli strains are responsible for host preference. T3Es that are unique to group II A. citrulli strains including Aave_2708 and Aave_2166 have been deleted individually, but these mutants still were unable to infect melon fruit tissue. It is possible that these genes must be deleted simultaneously to determine the role of these effectors in A. citrulli virulence and host specificity in cucurbit.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Dutta, B., Schneider, R.W., Robertson, C.W., and Walcott R.R. 2016. Embryo localization enhances the survival of Acidovorax citrulli in watermelon seeds. Phytopathology 106:330-338
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Jiang N., Lv, Q.Y., Xu, X., Cao, Y.S., Walcott, R.R., Li, J.Q., Luo, L.X. 2016. Induction of the viable but nonculturable state in Clavibacter michiganensis subsp. michiganensis and in planta resuscitation of the cells on tomato seedlings. Plant Pathology 65:826-836
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Zivanovic, M. and Walcott, R.R. 2017. Further characterization of genetically distinct groups of Acidovorax citrulli strains. Phytopathology 107:29-35
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Tian Y., Da, L., Zhao, L., Wu, J., Hu, B. and Walcott, R.R. 2016. Visual detection of Didymella bryoniae in cucurbit seeds using a loop-mediated isothermal amplification assay. European J. Plant Pathology doi:10.1007/s10658-016-0996-5
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Silva, G.M., Souza, R.M., Yan, L., Sales Junior, R., Medeiros, F.H.V. and Walcott, R.R. 2016. Strains of the Group I lineage of Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbitaceous crops, are predominant in Brazil. Phytopathology 106:1486-1494
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Eckshtain-Levi, N., Shkedy, D., Gershovits, M., DaSilva, G.M., Dafna, T.A., Walcott, R., Pupko, T., Burdman, S. 2016. Insights from the genome sequence of Acidovorax citrulli M6, a group I strain of the causal agent of bacterial fruit blotch of cucurbits. Frontiers in Microbiology 7:430 (doi: 10.3389/fmicb.2016.00430)
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