Progress 03/01/10 to 02/28/15
Outputs
Target Audience:1. The target audience for this reporting period were agricultural research personnel via regional and state meetings, sweetpotato growers via local meetings and one-on-one interactions in the field, extension agriculture personnel via local and regional meetings, and plant pathologists and students via regional and state meetings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Compilation of sweetpotato tip/end rot data collected during 2013 and 2014 is being completed, and will be statistically analyzed. Drafts of two journal articles are underway to provide descriptions of the culturable and non-culturable fungal and bacterial communities associated with sweetpotato tip/end rot at significant management periods throughout the growing season and after harvest. Upon completion of the greenhouse studies data collection, field and greenhouse analysis of the interaction between Macrophomina phaseolina and reniform nematode will also be compared, and potentially included in Extension publications for use by sweetpotato producers within the state. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Sweetpotato Tip/End rot Research: Studies are ongoing to define and evaluate the pathogen(s) associated with this disease problem that was severe and widespread in 2009, but can still be found at high levels following harvesting. An initial study (Phase 1) was established in 2009 to determine the causal agents associated with tip/end rot and completed in 2013. During 2012 it was determined that reniform nematode, Rotylenchulus reniformis, levels were increasing in many fields from a general survey and was suspected that they might form a disease complex resulting in greater tip/end rot in Mississippi. Research was established in 2013 to evaluate those interactions with the most common pathogen isolated, Macrophomina phaseolina and reniform nematode using different populations in the field and in the greenhouse (Phase 2). Phase 1 (Now Completed): Microbes were sampled from plant tissues over eight production stages, from seed stock potatoes used in bedding fields to produce slips, through transplanting of the slips into the production fields, until potatoes were stored up to 90 days post-harvest. A number of fungi, Macrophomina phaseolina, Aspergillus flavus, A. niger, A. tubingens, A. japonicus, and six species of Fusarium, were pathogenic in trials on disease-free sweetpotato root tissue. The relative occurrence of each of the identified pathogens was also analyzed using the multivariate analysis software PC-ORD (MjM Software, Inc.). Fusarium pathogenic species were the dominant pathogenic isolate recovered from plant tissues during early season sampling, but in harvest through post-harvest sampling, Macrophomina phaseolina became the dominant pathogenic fungi recovered from plant tissues. • Phase 2(ongoing): The purpose of this study is to define the relationship between reniform nematodes and pathogenic fungi that are associated with tip/end rot disease of sweetpotatoes. We will use data from year one of our ongoing study for 2013-14, funded by the USDA Specialty Crops Research Initiative grant, to clearly define the relationship between the organisms, categorize risks to sweet potato growers, and propose management strategies to minimize assessed risks. Year 1 data indicates that areas of high nematode populations show a decreased trends in average number of potatoes per plant, plant dry weights, and average length and width of root sizes. Prior to initiation of 2014 field research, data was collected on the densities of reniform (root knot) nematode, Mp levels based on past history of pathogen in selected fields and the consequential end rot development. In both 2013 and 2014 growing seasons, two fields with known histories of high nematode densities were sampled using plots of 1/1000 acre (4.05m2), with reniform density combinations as the treatments. Treatments within each field site were based on reniform counts including low range and high range. Twelve replicate plots per treatment were established per location. Data collection included destructive sampling of plants within each of the small plots at the following intervals: 1. Immediately before transplant 2. At harvest (90-120 days) 3. 60 days post-harvest 4. 90 days post-harvest 5. 120 days post-harvest. Soil samples of approximately one pint (0.47 L) were taken randomly throughout each of the small plots at the same intervals to determine nematode population densities. Plant tissues collected during destructive sampling were examined for nematode damage and fungi from samples are being cultured on PDA. Identified fungi will be recorded and a sample of each fungus and tissue sample will be stored in 15% glycerol at -80°C. Harvestable yield data will be collected for each plot at the end of the growth cycle when the field is harvested by the grower. • Greenhouse studies - Eight treatments were used for this experiment including 1. nontreated control, 2. Mp fungal control at 15 mL of cornmeal sand inoculum, 3-5. three levels of reniform nematodes at field plot average, 1/3 below average and 2/3 below average, and 6-8. the same levels of reniform nematode plus 15 mL of cornmeal sand inoculum containing Mp. Nematode level proposed here were based on previous sweetpotato-reniform research by Clark and Wright 1983. A randomized block design was employed with 8 replicate pots per treatment was to simulate field disease-nematode conditions. Based on initial soil sampling of five fields, a multi-factorial layout will be employed as in 2013 to test several combinations of nematode density and pathogenic fungi presence or absence. Tissue culture grown sweetpotato slips will be planted in pots of pasteurized ProMix BX to eliminate microbial interference and maintained at the recommended moisture content and temperature (Thompson et al., 2002). Destructive sampling will be performed as in field studies. Sweetpotato roots will be harvested and stored at 55° to 60° F (12.7° to 15.5° C) and 85 to 90 % humidity (Thompson et al., 2002). Plant tissues collected during destructive sampling will be examined for nematode damage and will be cultured on PDA. A portion of each fungus and tissue sample will also be stored for future use at -80°C. Summary of Sweetpotato Tip/End Rot Data - Year one of data collection indicated some numerical differences in plant weight, average number of potatoes produced, average length and width of potatoes, and small numerical differences in disease rating based on two types of treatments - low and high nematode populations. Data analysis through mid-season shows the significantly different nematode levels in the treatment plots as well as the numerical differences in average number and size of potatoes collected, with high nematode level plots producing somewhat fewer and smaller potatoes. Disease rating and plant weight differences between treatments indicate that there may be less plant growth and more disease incidence in plots with higher populations of nematodes. We expect to observe more marked differences and a greater separation of data between treatments after data collection for Year 2 has been completed. • Soybean Charcoal Rot Disease Research - Throughout April, May, and June 2014, work continued on isolation of DNA from soybean tissues collected in 2012 and 2013 growing seasons. Amplification with ITS primers ITS1f and ITS4 to select fungal DNA sequences is ongoing. Primers labeled with unique barcodes necessary for Illumina sequencing of total DNA have been obtained and techniques for preparing the samples for MiSeq processing have been established. Identification of culturable fungal isolates has been completed from the 2013 field samples. The most commonly occurring species are Phoma sp., Penicillium chryseogenum, Diaporthe phaseolorum, Fusarium sp., Aspergillus flavus, Diaporthe sojae, Fusarium oxysporum, Trichoderma reesei, Hypocrea lixii, and Alternaria alternata. Continuing work includes the extraction of DNA from whole soybean plant tissues (plant DNA and DNA of associated microorganisms), followed by amplification of the ITS1 gene region of any fungi associated with these tissues. The reverse ITS primers (ITS4) used for this procedure contain unique barcodes, which will allow separation and distinction of any non-culturable species that are present within the plant tissue via Illumina whole-community sequencing. DNA extraction is currently underway and PCR required for identification of fungi using Illumina sequencing of ITS DNA. • Aspergillus flavus Research in Corn - One publication was accepted this year, "Monitoring MVOC Profiles over Time from Isolates of Aspergillus flavus using SPME GC-MS", authored by Dongdi Sun, Alicia Wood-Jones, Wenshuang Wang, Chris Vanlangenberg, David Jones, Julie Gower, Patrice Simmons, Richard Baird, and Todd Mlsna. Active research in this area has been completed, upon the graduation of two PhD students (Sun and Wood-Jones), who were tasked with completing this work.
Publications
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: The target audience for this reporting period were agricultural research personnel via regional and state meetings, sweetpotato growers via local meetings and one-on-one interactions in the field, extension agriculture personnel via local and regional meetings, and plant pathologists and students via regional and state meetings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? Compilation of sweetpotato tip/end rot data collected during 2013 and 2014 is being completed, and will be statistically analyzed. Drafts of two journal articles are underway to provide descriptions of the culturable and non-culturable fungal and bacterial communities associated with sweetpotato tip/end rot at significant management periods throughout the growing season and after harvest. Upon completion of the greenhouse studies data collection, field and greenhouse analysis of the interaction between Macrophomina phaseolina and reniform nematode will also be compared, and potentially included in Extension publications for use by sweetpotato producers within the state. What do you plan to do during the next reporting period to accomplish the goals? During the next reporting period, data compilation, greenhouse trials, and statistical analyses of data will be completed to provide researchers and growers alike with a comprehensive picture of the microbial community associated with sweetpotato tip/end rot, and with charcoal rot of soybean.
Impacts
What was accomplished under these goals?
Sweetpotato Tip/End rot Research: Studies are ongoing to define and evaluate the pathogen(s) associated with this disease problem that was severe and widespread in 2009, but can still be found at high levels following harvesting. An initial study (Phase 1) was established in 2009 to determine the causal agents associated with tip/end rot and completed in 2013. During 2012 it was determined that reniform nematode, Rotylenchulus reniformis, levels were increasing in many fields from a general survey and was suspected that they might form a disease complex resulting in greater tip/end rot in Mississippi. Research was established in 2013 to evaluate those interactions with the most common pathogen isolated, Macrophomina phaseolina and reniform nematode using different populations in the field and in the greenhouse (Phase 2). Phase 1 (Now Completed): Microbes were sampled from plant tissues over eight production stages, from seed stock potatoes used in bedding fields to produce slips, through transplanting of the slips into the production fields, until potatoes were stored up to 90 days post-harvest. A number of fungi, Macrophomina phaseolina, Aspergillus flavus, A. niger, A. tubingens, A. japonicus, and six species of Fusarium, were pathogenic in trials on disease-free sweetpotato root tissue. The relative occurrence of each of the identified pathogens was also analyzed using the multivariate analysis software PC-ORD (MjM Software, Inc.). Fusarium pathogenic species were the dominant pathogenic isolate recovered from plant tissues during early season sampling, but in harvest through post-harvest sampling, Macrophomina phaseolina became the dominant pathogenic fungi recovered from plant tissues. Phase 2(ongoing): The purpose of this study is to define the relationship between reniform nematodes and pathogenic fungi that are associated with tip/end rot disease of sweetpotatoes. We will use data from year one of our ongoing study for 2013-14, funded by the USDA Specialty Crops Research Initiative grant, to clearly define the relationship between the organisms, categorize risks to sweet potato growers, and propose management strategies to minimize assessed risks. Year 1 data indicates that areas of high nematode populations show a decreased trends in average number of potatoes per plant, plant dry weights, and average length and width of root sizes. Prior to initiation of 2014 field research, data was collected on the densities of reniform (root knot) nematode, Mp levels based on past history of pathogen in selected fields and the consequential end rot development. In both 2013 and 2014 growing seasons, two fields with known histories of high nematode densities were sampled using plots of 1/1000 acre (4.05m2), with reniform density combinations as the treatments. Treatments within each field site were based on reniform counts including low range and high range. Twelve replicate plots per treatment were established per location. Data collection included destructive sampling of plants within each of the small plots at the following intervals: 1. Immediately before transplant 2. At harvest (90-120 days) 3. 60 days post-harvest 4. 90 days post-harvest 5. 120 days post-harvest. Soil samples of approximately one pint (0.47 L) were taken randomly throughout each of the small plots at the same intervals to determine nematode population densities. Plant tissues collected during destructive sampling were examined for nematode damage and fungi from samples are being cultured on PDA. Identified fungi will be recorded and a sample of each fungus and tissue sample will be stored in 15% glycerol at -80°C. Harvestable yield data will be collected for each plot at the end of the growth cycle when the field is harvested by the grower. Greenhouse studies – Eight treatments were used for this experiment including 1. nontreated control, 2. Mp fungal control at 15 mL of cornmeal sand inoculum, 3-5. three levels of reniform nematodes at field plot average, 1/3 below average and 2/3 below average, and 6-8. the same levels of reniform nematode plus 15 mL of cornmeal sand inoculum containing Mp. Nematode level proposed here were based on previous sweetpotato-reniform research by Clark and Wright 1983. A randomized block design was employed with 8 replicate pots per treatment was to simulate field disease-nematode conditions. Based on initial soil sampling of five fields, a multi-factorial layout will be employed as in 2013 to test several combinations of nematode density and pathogenic fungi presence or absence. Tissue culture grown sweetpotato slips will be planted in pots of pasteurized ProMix BX to eliminate microbial interference and maintained at the recommended moisture content and temperature (Thompson et al., 2002). Destructive sampling will be performed as in field studies. Sweetpotato roots will be harvested and stored at 55° to 60° F (12.7° to 15.5° C) and 85 to 90 % humidity (Thompson et al., 2002). Plant tissues collected during destructive sampling will be examined for nematode damage and will be cultured on PDA. A portion of each fungus and tissue sample will also be stored for future use at -80°C. Summary of Sweetpotato Tip/End Rot Data - Year one of data collection indicated some numerical differences in plant weight, average number of potatoes produced, average length and width of potatoes, and small numerical differences in disease rating based on two types of treatments – low and high nematode populations. Data analysis through mid-season shows the significantly different nematode levels in the treatment plots as well as the numerical differences in average number and size of potatoes collected, with high nematode level plots producing somewhat fewer and smaller potatoes. Disease rating and plant weight differences between treatments indicate that there may be less plant growth and more disease incidence in plots with higher populations of nematodes. We expect to observe more marked differences and a greater separation of data between treatments after data collection for Year 2 has been completed. Soybean Charcoal Rot Disease Research - Throughout April, May, and June 2014, work continued on isolation of DNA from soybean tissues collected in 2012 and 2013 growing seasons. Amplification with ITS primers ITS1f and ITS4 to select fungal DNA sequences is ongoing. Primers labeled with unique barcodes necessary for Illumina sequencing of total DNA have been obtained and techniques for preparing the samples for MiSeq processing have been established. Identification of culturable fungal isolates has been completed from the 2013 field samples. The most commonly occurring species are Phoma sp., Penicillium chryseogenum, Diaporthe phaseolorum, Fusarium sp., Aspergillus flavus, Diaporthe sojae, Fusarium oxysporum, Trichoderma reesei, Hypocrea lixii, and Alternaria alternata. Continuing work includes the extraction of DNA from whole soybean plant tissues (plant DNA and DNA of associated microorganisms), followed by amplification of the ITS1 gene region of any fungi associated with these tissues. The reverse ITS primers (ITS4) used for this procedure contain unique barcodes, which will allow separation and distinction of any non-culturable species that are present within the plant tissue via Illumina whole-community sequencing. DNA extraction is currently underway and PCR required for identification of fungi using Illumina sequencing of ITS DNA. Aspergillus flavus Research in Corn - One publication was accepted this year, "Monitoring MVOC Profiles over Time from Isolates of Aspergillus flavus using SPME GC-MS", authored by Dongdi Sun, Alicia Wood-Jones, Wenshuang Wang, Chris Vanlangenberg, David Jones, Julie Gower, Patrice Simmons, Richard Baird, and Todd Mlsna. Active research in this area has been completed, upon the graduation of two PhD students (Sun and Wood-Jones), who were tasked with completing this work.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Sun, Dongdi, Alicia Wood-Jones, Wenshuang Wang, Chris Vanlangenberg, David Jones, Julie Gower, Patrice Simmons, Richard E. Baird, Todd E. Mlsna. 2014. Monitoring MVOC Profiles over Time from Isolates of Aspergillus flavus using SPME GC-MS. Journal of Agricultural Chemistry and Environment. Volume 3, pp. 48-63.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Stokes, C.E., R.A. Arancibia, and R.E. Baird. Tip/End Rot Update. Mississippi Sweetpotato Producers Advisory Meeting. February 20, 2014
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Stokes, C.E., and R.E. Baird. Tip/End Rot Update. Mississippi Sweetpotato Growers Meeting. August 20, 2014
|
Progress 01/01/13 to 09/30/13
Outputs
Target Audience: The target audience for this reporting period were agricultural research personnel via regional and national meetings, sweetpotato growers via local meetings and one-on-one interactions in the field, extension agriculture personnel via local and regional meetings, and plant pathologists and studens via regional and national meetings. Changes/Problems: In 2012, many fields with higher levels of sweetpotato tip/end rot were also found to contain high levels of reniform nematode not present in the past. Therefore, field and greenhouse trials were established after the discovery of a potential interaction between the commonly isolated pathogen Macrophomina phaseolina and the reniform nematode Rotylenchulus reniformis. The field and greenhouse trials established in 2013 are currently ongoing. In two fields known to have a history of tip/end rot, 1/1000 acre plots were established in areas surveyed and found to have either relatively high (2659 - 10,843)or low (63-1385) populations of reniform nematodes (treatments). Two rows were set aside within each plot for determination of harvested yield, and plants were randomly selected from the remaining rows for destructive sampling throughout the growing season. Stand count, root disease evaluation, harvestable yield, and number and size of potatoes produced were evaluated in each plot throughout the season. Data is currently being analyzed for the 2013 growing season, and will be collected during the storage period (60-90 days) for the harvested roots. Greenhouse studies are examining interactions of varying levels of M. phaseolina with and without the presence of reniform nematodes. Half of the eight replicates for each treatment will be destructively sampled at the mid-point (60 days) of the growing cycle to examine root disease, root production, nematode population count adjacent to plant, and presence of M. phaseolina within the plant tissues. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? Services: Consulting with the following sweetpotato production companies – Bailey Family Farms, Topashaw Farms, Penick Produce, N&W Farms, Langley Farms, C&W Farms, Alexander Farms LLC. Corn aflatoxin data is being provided to USDA-ARS for resistance research efforts and to the Corn Growers Commission for dissemination of results found during the investigations completed in 2013. Events: Participated in the 2013 Producers Advisory Council (Verona, MS); 2013 Spring Sweetpotato Growers Meeting (Vardaman, MS); 2013 Sweetpotato Field Day (Pontotoc, MS); 2013 National Sweetpotato Growers Council annual meeting (Orlando, FL). Activities: Surveyed fields with a history of end rot and reniform nematode damage for Bailey Family Farms, Topashaw Farms, and Penick Produce. Offered nematode survey services to all participants at Producers Advisory Council and Sweetpotato Growers meetings in February 2013. What do you plan to do during the next reporting period to accomplish the goals? Research effortswill continueon the interaction of Macrophomina phaseolina and reniform nematode in a disease complexof tip/end rot symptoms in sweetpotato. Greenhouse studiesand field trials will be replicated in 2014in fields located in north Mississippithat have a history of tip/end rot and pockets of high reniform nematode populations. For soybean endophyte biocontrol study, fungal and bacterialisolates obtained from symptomatic and asymptomatic soybean plants fromselect locations in 2013will be sequenced for confimation of theiridentities. Tissues collected from the plants will also be subjected to DNA extraction and whole-community sequencing through Illumina pyrosequencing.
Impacts
What was accomplished under these goals?
1-2. Work was completed on objective 1, with 11 common pathogenic fungi occurring throughout the growing season, in up to 70% of isolations. Further pathogen screening is needed, particularly with the six common Fusariumspecies that were isolated throughout all sampling periods. Bacterial identifications were completed from representative samples of 7,509 isolates, with three bacteria, Bacillus spp., Lysobacter enzymogenes, and Paenibacillus lentimorbus, occurring across the eight production stages.Twenty-eight additional taxa were identified by both fatty acid profile analysis and 16S sequencing. None were found to be pathogenic when screened on non-infected sweetpotato root tissue. Someidentified bacteria, Photorhabdus luminescens, Paenibacillus lentimorbus, and Bacilluscereusare notable for acting as biocontrol agents in certain disease complexes. Multivariate analysis of the bacterial populationindicated a diverse population of microorganisms throughout the growing season, with the closest correlationsoccurring between sampling periods 7a, 8a,7, and 8, all post-harvest storage root sampling points over two growing seasons.Results for fungal diversity analysisshow a wide distribution of species across many management periods, but the harvest and post-harvest sampling periods show the greatest species richness over both years. Macrophomina phaseolina, Aspergillus flavus, A. niger, A. tubingens, A. japonicus, and six species of Fusarium, were pathogenic in trials on disease-free sweetpotato root tissue. In addition, F. oxysporum and F. solani consistently produced necrotic lesions in root tissue. These two species accounted for nearly 70% of the overall isolates from early season seed stock and bedding plant samples. Microbial populations in post-harvest tissues also differed in relative abundance from prior sampling dates. Macrophomina phaseolina increased to 27% occurrence and F. oxysporum and F. solani decreased to 15.5% isolation frequencies between 60 and 90 days post-harvest. During this work, a possible interaction between the fungal disease component, Macrophomina phaseolina, and high populations of reniform nematode, Rotylenchulus reniformis, was discovered. The current work effort aims to determine how these two organisms correlate in production of tip/end rot symptoms. 2 - 5. Corn Aflatoxin work: A specifically designed oligonucleotide, dual-labeled florescent probe (OMG3) to amplify DNA within a specific gene target aflP (within the aflatoxin biosynthetic pathway) was used. A standard curve was developed based on known DNA concentrations, and the curve could then be used to evaluate comparing resistance and susceptible genotypes. The QPCR method and the novel OMG3 probe were tested. Both in vivo and in situ procedures were developed and refined. The toothpick inoculation method was used to artificially inoculate maize stems in the vegetative stage. This method was 91% consistent for infecting maize plants however inoculation must occur during the vegetative V5 stage of growth to avoid mechanical tissue damage. Only lesion length data in 4 out of the 7 greenhouse trials showed significantly greater measured lengths in resistant lines than in susceptible lines. Based on this data, additional research needs to focus on refining tissue-sampling methods; sampling in 0.5 increments in lengths up to 3 cm or even increasing length of time for tissue sampling following inoculation. Procedural changes in sampling procedures may provide more useful data when evaluating maize genotypes. Solid phase microextraction gas chromatography – mass spectrometry was used to determine the predominant microbial volatile organic compounds (MVOC’s) extracted by from both aflatoxigenic and non-aflatoxigenic strains. The major MVOC’s from both strains were alcohols, ketones and hydrocarbons. Dimethylsulfide and 2-heptanol were key MVOC biomarkers and were produced only by the aflatoxigenic strain of A. flavus and distinguished the two strains. Charcoal rot in soybean work: Symptomatic and asymptomatic soybean plants in a field known to be affected by charcoal rot were collected twice in 2013. Soil samples from root zone of each of the twelve plants were collected and sent to the Mississippi State University Extension Plant Disease and Nematode Diagnostic Laboratory for analysis. Plants were surface disinfected and split longitudinally with half of each plant used for isolation of fungal endophytes and half used for isolation of bacterial endophytes. Bacteria were isolated using an enrichment protocol with the majority retained for DNA sequencing and a fraction of each sample cultured on two media in three different environments (aerobic, microaerobic and anaerobic). Some of the bacteria from these cultures have been collected with representative colonies selected for storage at -80C and the remaining bacteria washed from the plates for DNA extraction. Some of the microaerobic and anaerobic cultures remain in incubation and will be collected when adequate growth is observed. Identification of bacteria will be accomplished using Illumina sequencing of 16S DNA after all cultures are harvested. Fungi were isolated from stem, root, and leaf tissue of each plant using two selective growth media. Sections (1-cm) were aseptically excised and placed onto each media plate, three replicates of each, and incubated in the same environments used for the bacteria. Fungal growth from each plant tissue piece was subcultured onto individual small media plates, and these were grouped visually. To date 4-5 morphotypes of Fusarium spp., Macrophomina phaseolina, and at least one Trichoderma sp. have been identified using traditional techniques. Total of 25% of isolates from each morphological group is being used for DNA extraction to confirm the identity of each group of isolates by ITS sequencing.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
Stokes, C.E., R.A. Arancibia, and R.E. Baird. Diversity and density indices of the pathogenic microbial community present in tip/end rot disease of sweetpotato. National Sweetpotato Growers Council/ American Society for Horticultural Science annual meeting.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
Stokes, C.E., R.A. Arancibia, and R.E. Baird. Biodiversity of the fungal community present in sweetpotato (Ipomoea batatas L. Lam). Mycological Society of America annual meeting.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
Stokes, C.E., R.A. Arancibia, and R.E. Baird. Microbial disease complex of sweetpotato (Ipomoea batatas L. Lam) tip/end rot. American Phytopathological Society annual meeting.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
Stokes, C.E., R.A. Arancibia, and R.E. Baird. Fungi and bacteria isolated from tip/end rot diseased sweetpotatoes in Mississippi: Community analysis and pathogen identification. American Phytopathological Society Southern Division annual meeting
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
Stokes, C.E., and R.E. Baird. Tip/end rot research update. Mississippi Sweetpotato Growers Spring 2013 meeting.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
Stokes, C.E., R.A. Arancibia, and R.E. Baird. Tip/end rot update. Mississippi Sweetpotato Growers 2013 Field Day.
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2013
Citation:
Wood-Jones, A. 2013. Evaluation of quantitative polymerase reaction and microbial volatile organic compounds to determine resistance to Aspergillus flavus in maize.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2014
Citation:
Wood-Jones, A. D. Sun, T.E. Mlsna, W. Wang, C. Vanlangenberg, P.D. Jones, and R.E. Baird. 2013. Discrimination of aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus based on volatile metabolic profiles using SPME GCMS and multivariate analysis.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Corn aflatoxin research: Over the last year, quanitative real-time PCR probes for identification of Aspergillus flavus toxigenic isolates were developed for quantifying fungal genomic DNA. Research focused on specificity of probes and their development for biomass studies, which included evaluating varieties for resistance to this mycotoxin-forming fungus. Specifically, the research developed a possible method to determine resistance in corn hybrids (seedlings) by quantification of fungal genomic DNA using quantitative real-time PCR. The primers and the novel TaqMan probe developed in this laboratory can distinguish the aflatoxigenic strain NRRL 3357 currently used in field resistance trials by the USDA-ARS-CHPRRU from the Afla-Guard strains. Sweetpotato tip/end rot research: Sweetpotato research was initiated in 2010 to first determine associated microbes at different stages of plant development and storage. For the two year sampling period, approximately 7,500 bacterial isolates and 7,300 fungal isolates were collected. After initial species grouping, approximately 300-350 fungal isolates and 350-400 bacterial isolates were selected for sequence analyses. FAME analysis through Sherlock GC software from MIDI, Inc. included the bacterial species Bacillus cereus, Bacillus subtilis, Lysobacter enzymogenes, Paenibacillus lentimorbus, Photorhabdus luminescens, and Stenotrophomonas maltophilia, among others. Morphological grouping of fungi indicated that that majority of isolates were Macrophomina phaseolina, Aspergillus spp., Trichoderma spp., and a large number of Fusarium spp. To confirm identity of isolates, the bacterial 16S rDNA region was amplified using the 8F and 1492R primers. From fungi, the ITS region was amplified with primers ITS1 and ITS4. Isolates with confirmed identities include F. solani (14), F. oxysporum (13), Fusarium spp. (8), Phoma epicoccina (5), Nectria haematococca (6), Mucor circinelloides (1), Penicillium oxalicum (3), B.cereus (14), B.subtilis (8), B.megaterium (9), P.luminescens (9), E. hormachei (6), P.lentimorbus (9), and S.maltophilia (9). Fungal isolates were found in all tissue types that were sampled, and there was no statistical difference in the frequency of isolate occurrence between tissue types (parenchyma, stem end, root end). Pathogenicity screening studies have indicated no tip/end rot production by bacterial isolates. Fungal isolates found to have a positive pathogenicity are Fusarium isolates (BF, BB, DP, N), and Macrophomina phaseolina isolates (N1, B4, J3, T2, G5, AP, AA). Of these, only Fusarium BF and DP and M.phaseolina AA show severe tissue necrosis as in tip/end rot disease. Forest ecosystem research: Investigations on fungal systematics of pathogenic fungi of agricultural crops/forest ecosystems and molecular sampling of mycorrhizal fungi affect by enviromental influences was conducted using traditional and molecular approaches. PARTICIPANTS: For the corn studies Alicia Wood-Jones is conducting the reseach and is at the end stages of the project for a Ph.D. Dr. Beth Stokes is the post-doctoral associate conducting the daily research on the sweetpotato project. I am the overall project leader for both funded studies. The latter project is being conducted in support with a regional effort especially from Lousiana State University personnel. TARGET AUDIENCES: Scientists in related field. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Corn aflatoxin research: The primers and the novel TaqMan probe developed in this laboratory can distinguish the aflatoxigenic strain NRRL 3357 currently used in field resistance trials by the USDA-ARS-CHPRRU from the Afla-Guard strains. Data suggests from these greenhouse studies that fungal densities found within inoculated maize seedlings at the V5 stage can possibly be correlated with susceptibility to the fungus only if greenhouse conditions are adequate for fungal infection. However, impact of the results may be dependent on environmental conditions in the greenhouse with additional testing needing to refine conditions. Sweetpotato tip/end rot research: Relative abundance within bacterial communities appears to shift between the growing cycle and harvest, with bacterial isolates recovered from harvested roots at 60 and 90 days post-harvest showing the greatest abundance of species. Bacillus spp., Photorhabdus luminescens, and Paenibacillus lentimorbus account for more than 50% of total identifications throughout sampling, with Bacillus spp. decreasing in abundance post-harvest as other species became more abundant. Interestingly, the frequently occurring P. luminescens is most often associated with the beneficial nematode Heterorhabditis bacteriophora, which infects a variety of insect hosts with developmental stages in soil. Bacillus cereus strains are used in the biological control of fungal diseases in crops, such as damping off in alfalfa and early leafspot of peanut, and may be used to control the rootknot nematode Meloidogyne javanica. P. lentimorbus has also been reported to act as a biological control for early blight disease in tomato, by inducing host resistance gene expression and inhibiting the growth of Alternaria solani. These ubiquitous soil and water bacteria do not appear to be pathogenic to sweetpotato root tissues according to literature and our own pathogenicity trials. Fungal communities also showed variation in relative abundance throughout the growing season, with the seed stock and bedding field slips showing greater variation in the fungal community than was seen in 60 and 90 day post-harvest samples. Fusarium spp. decreased significantly from early season samples to post-harvest samples, with the fewest Fusarium spp. isolates recovered at 90 days post-harvest. From pathogenicity trials, three fungal isolates have been found to have severe pathogenic activity Fusarium BF, Fusarium DP, and Macrophomina phaseolina AA. These three isolates have shown repeated ability to completely degrade slices of disease-free sweetpotato storage root, and are currently being tested for their impact on whole plants in a controlled environment. By determining the pathogenic activity of individual strains of known pathogens on whole plants, we can improve recommendations to sweetpotato growers for management practices to limit or eliminate tip/end rot in their crop.
Publications
- Stokes, C.E., S.W. Woolfolk, R.A. Arancibia, and R.E. Baird. 2012. Diversity, Densities, and Distribution of Microbial Communities in Sweetpotato End/Tip Rot Diseases. National Sweetpotato Collaborators Group 2011 Progress Report, NSCG Annual Meeting, Birmingham, Alabama.(p 23-24)
- Wang, X., P. Wadl, A. Wood-Jones, G. Windham, R. N. Trigiano, M. Scruggs, C. Pilgrim, and R. Baird. 2012. Characterization of expressed sequence tag-derived simple sequence repeat markers for Aspergillus flavus: Emphasis on variability of isolates from the southern United States. Mycopathologia 174: 371-382.
- Clark, C., R. Arancibia, R. Baird, B. Burdine, A. Villordon, J. Schultheis, M. Shankle, Z. Pesic-van Esbroeck. 2012. Objective 3 Group Update: Etiology and Biology of Emerging End Rot Diseases. USDA: Specialty Crops Research Initiative Sweet Potato Project Group Meeting, February 3, 2012, Birmingham, Alabama (Abstract).
- Wood-Jones, A.K., Sun, D., Mlsna, T. and Baird, R.E. 2011. Evaluating the use of solid-phase microextraction to detect aflatoxin-producing isolates of the fungus Aspergillus flavus. American Phytopathological Society and the International Association for Plant Protection Services, National meeting. Honolulu, HI. 2011. (Abstract)
- Xinwang, W., P. A. Wadl, A. Wood-Jones, G. Windham, R. N. Trigiano, C. Pilgrim, M. Scruggs, and R. Baird. 2012. Determination of isolate variability of United States isolates of Aspergillus flavus based on simple sequence repeats: Emphasis on mid-south samples. Mycopathalogia (In review)
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The most commonly identified fungal species were Fusarium spp., Botryodiplodia theobromae, Macrophomina spp., and Aspergillus niger. Between management period 7 and 8, when end-rot symptoms become visible, the distribution and total number of fungal species changed significantly. Botryodiplodia theobromae, Fusarium spp. and Macrophomina spp. increased in number in the Site 1 field, while the dominant Aspergillus niger from the previous sampling period was not detected. Storage roots taken from the Site 2 field showed an increased occurrence of Fusarium spp. at management period 8. We also detected a greater variety of isolates in samples from management period 8 (Site 1 n=36; Site 2 n=25) than we did in samples from management period 7 (Site 1 n=16; Site 2 n=15). Fungal isolates were found in all tissue types that were sampled, and there was no statistical difference in the frequency of isolate occurrence between tissue types (parenchyma, stem end, root end). PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Other scientists working on sweetpotato tip/end rot research or growers interested in update of progress to find the cause(s). PROJECT MODIFICATIONS: Not relevant to this project. PARTICIPANTS: The project consists of of PI who oversees all operations and ensures completion of objectives. This includes monthly updates from two post-docs and consults when changes needed to be made. Two post-docs work on the daily operation of the project with one primarily responsible for field and isolation work. The other post doc supports and assists with field and pathogenicity screening from preliminary tests in the incubator to greenhouse studies. Last year one post-doc will remain and assist with identification of bacteria and fungi using PCR, fatty acids determinations and conventional microscopic identification (fungi primarily) or fermentation confirmations with bacteria. Two student workers do daily media preparation, assist with isolations, and will help with any aspects of incubator and greenhouse pathogenicity screening. When appropriate one student with assist with laboratory identification research (PCR and fatty acids). TARGET AUDIENCES: Target audience will include plant pathology researchers from the southern region so they can initiate field screening with known pathogens in hopes of developing control measures. Other personnel needing the information are consultants and horticultural scientists who will be looking into physiological changes in plants in presence of these fungi/bacteria. All data will then be forward to county extension personnel in appropriate sweetpotato counties and growers directly. Any recommendations from the work will be passed to the growers but this is more longterm. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
: When compared to fungal isolate enumeration data from the baseline samples taken in management period 1, we see a difference in the identified isolates. The most common isolates continue to be identified as Fusarium spp., Aspergillus spp., and Macrophomina spp. The distribution, however, is significantly different to what we see in later samples. There are more isolates found in each site (Site 1 n=47; Site 2 n= 39), and the number of occurrences of each isolate is much less. Bacteria isolated from samples included many common soil bacteria, including Bacillus cereus, Bacillus subtilis, Paenibcillus spp., Stenotrophomonas maltophilia and Micrococcus spp. These bacteria in particular have been reported as endophytes of sweetpotato (Stenotrophomonas maltophilia) or as pathogens capable of causing soft rots in sweetpotato and other root vegetables (B.cereus, B.subtilis) (Khan and Doty 2009, Narayanasamy 2006). These were identified as early as seed stock samples and bedding field plant samples. Comparison of these results with subsequent sampling points is ongoing. Isolates are being tested for pathogenicity by inoculation of disease-free slices of sweet potato storage root tissue with 2-mm slices of fungal or bacterial isolate on growth media. We expect to examine 100-150 different isolates of fungi and bacteria for pathogenic characteristics. Trials conducted so far have produced several potential pathogens, including Fusarium BF and Fusarium AU. Isolates of fungi and bacteria that show pathogenic traits on sweet potato parenchyma tissue are being subjected to greenhouse plant trials to determine field-scale effects of individual isolates. Disease-free slips are being cultivated in a controlled environment, as shown in Figure 11, and will be inoculated with one of the previously mentioned pathogens at the time of transplant. Replicate groups are composed of four plants each and an uninoculated control group is being monitored simultaneously. Storage roots are being allowed to mature to a harvestable size, and tissue samples equivalent to those removed from the field study will be taken throughout the growth cycle. The collected tissue samples will be examined visually and by culturing for the presence of the introduced pathogen to determine at which point in the growth cycle the pathogen begins to invade the sweet potato tissues. Furthermore, to evaluate if the putative pathogen(s) is capable of infecting a plant under field conditions, any isolates determined to be pathogenic to both storage root tissue and intact sweet potato plants will be selected for field-level tests at the Pontotoc MAFES station.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Several projects are ongoing and will be continued or be initiated from 2005-2010 CREES project. 1) Continue the research on the biology and epidemiology of the charcoal rot pathogen. During last four years and present, data is being collected and analyzed comparing visible symptom severity and percent root infections to nutriental changes in plants and toxin production. Specifically, the work is being conducted to understand disease initiation and establishment of the charcoal rot pathogen, Macrophomina phaseolina as impacted by the parameters discussed above. In 2010 and presently, data from last several years of research is being analyzed to determine any correlations occurred with the above parameters; 2) In 2010, primers for identification of Aspergillus flavus toxagenic isolates were developed for use in real-time PCR. Research focused on specificity of primers and their development for biomass studies. Research will continue to focus on evaluating varieties for resistance to this mycotoxin forming fungus. Specifically, the research will continue to develop or refine methods to determine resistance in corn hybrids (seedlings) by quantification of DNA using Real-Time PCR; 3) sweetpotato research was initiated in 2010 to first determine associated microbes at different stages of plant development and storage. The data from these researchs are being used to conduct pathogenicity screening of organisms of interest; 4)additional research was continued to the potential of associate microflora from fire ant mounds for their putative control of the ants. and 5) Investigations on fungal systematics of pathogenic fungi of agricultural crops/forest ecosystems and molecular sampling of mycorrhizal fungi affect by enviromental influences is being conducted using traditional and molecular approaches. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Researchers and farmer clientele. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Corn Resistance Research: From our results in 2090-2010, it appears that seedlings can be evaluated for resistance work looking at fungal biomass in seedling tissues as an indication of resistance compared to current field research, requiring full harvest. Use of these newly developed methods can save years of variety testing over time resulting in hundreds of additional varieties being tested during same time period. Charcoal Rot Research: Plant Physiology Studies: Field plots: Funding was not available in 2010. Therefore, data was compiled and is currently being analyzed from several field seasons. The analyzes will show growers if specific nutrients can be a key factor in increased charcoal rot disease. Toxin research: Data still being collected but no funds to complete this work from last field season. Results from toxin studies will enable us to understand disease progression and possibly look at resistance breeding based on toxin levels in plants and could be used for resistance breeding. SSR molecular studies: SSR primers are now available for evaluating isolate genetic variability for better understanding of the pathogen pathogenicity potential in different regions of the country. Isolates from different regions continue to be compared with other complete data from 2009. Sweetpotato end/tip rot pathogen continues to be conducted. Approximately 2400 isolates of fungi and 2500 isolates of bacteria were collected from sampling period 1-6. Samples from period 7 are currently being processed. Identification of bacteria and fungi is currently under way. Several fungal genera which tentatively have been identified included Fusarium (several species), Candida, Curvularia, Cercosporella, Phoma-like, Aspergillus, and Macrophomina. Following identification, representative isolates of each taxon (fungi and bacteria) will be stored in -80C as sweetpotato end/tip rot study culture collections for other scientist to use for their research. Pathogencity screening will be conducted in 2011 and until further results from that research is conducted no other comments on this project can be made. Additional research on taxonomic of plant pathogenic (eg. Pestalotiopsis spp.) or ectomycorrhizal fungi (eg. stiptate hydnums) is being conducted. Results and subsquent monographs from these projects can be used by diagnosticians and other scientists. At least one monograph is expected to be completed in 2011.
Publications
- Sun, D., Wood-Jones, A., Milsna, T. and R. Baird. 2010. Volatile organic compounds unique to corn infested with Aspergillus flavus. Southeast/Southwest Regional ACS meeting. New Orleans, LA. (Abstract)
- Baird, R., P. Wadl, T. Rinehart, H. Abbas, T. Shier, and R. Trigiano. 2010. SSR marker for genetic comparison of Macrophomina phaseolina isolates from different states and hosts throughout the continental United States. Mycopathologia 170:169-180.
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