Duration: 10/01/1999 to 09/30/2004

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Ergot of grain sorghum caused by Claviceps africana is a new and potentially devastating disease emerging in the United States. The ergot epidemic in the Americas began in Brazil in 1995 and by October of 1997 had spread as far north as Nebraska. Very little is known about this pathogen or host-plant traits that might influence the infection cycle. Given this threat to U.S. sorghum production, a cooperative and integrated ergot research effort is needed that incorporates plant pathologists, biologists and geneticists across the Central Great Plains. The project described in this proposal will address this need by focusing on research questions concerning ergot biology and disease management strategies. Research on ergot biology will focus on questions relating to epidemiology, taxonomy, and alternate hosts of the pathogen. Research on disease management will focus on integrated ergot control strategies including cultural practices, chemical application, and host-plant mechanisms that contribute to disease resistance or ergot-avoidance. The development of this committee will help to eliminate unnecessary duplication of effort and will facilitate cooperative exchange of information and ideas.

Justification:

Sorghum (Sorghum bicolor (L) Moench) is the worlds fifth most important cereal crop with over 100 million acres of production each year. Sorghum is also an important feed grain in the U.S., which annually produces over 10 million acres of sorghum. Most of the sorghum production in the U.S. is under dryland conditions in the Central Great Plains. Bennett et al. (1990) in their book, "Modern Grain Sorghum Production," characterize grain sorghum as a "wonder crop" of semi-arid and arid agriculture. They note that production of grain sorghum has been revolutionized in the past 40 years and predicted that grain sorghum acreage will expand in response to the increasing yield potential of the crop.

Claviceps africana is a fungal pathogen that infects unfertilized ovaries (Bandyopadhyay et.al. 1996). Any condition that reduces male fertility of sorghum predisposes the crop to infection by C. africana which is capable of rapid infection and dispersal. There are two Claviceps species that cause ergot of sorghum (Bandyopadhyay et al. 1996). Sorghum ergot was first identified in India in 1917 (C. sorghi) and Kenya (C. africana) in 1924. Sorghum ergot was considered an Old World disease caused by a single species, C. sorghi, until 1991 when the African species was separately described as C. africana (Frederickson and Mantle 1988; Frederickson et al. 1991). C. africana has recently caused an ergot epidemic that has spread through most of the sorghum-producing areas in North and South America, and Australia (Arias-Rivas et al. 1997). The ergot epidemic in the Americas began in Brazil in 1995. By 1996, ergot had spread across most of South American and to Australia. Ergot was identified in Mexico and the Caribbean in February of 1997 and by late March was found in southern Texas. The disease spread through the U.S. during the summer of 1997 and by October had spread as far north as Nebraska. Ergot has become an endemic problem in most sorghum producing areas in the U.S. and the world.

For years ergot has been a problem in Africa and India and it has now become endemic in the U.S. and is a growing problem (Bandyopadhyay et al. 1996). Seed production fields are particularly at risk. The seed parents used to produce hybrid seed are male-sterile, and male- sterility predisposes florets to ergot infection. Intensive fungicide treatment and careful management are required to limit this threat; however, little information is available to determine best management and control strategies. Ergot may also be a problem in grain production fields where intensive management may not be feasible. Cool, wet weather during the flowering period could reduce pollen viability and lead to conditions that are favorable to wide-spread ergot infection (Brooking, I.R. 1979; McLaren and Wehner 1992). The development and use of sorghum hybrids that remain highly fertile under cool, wet conditions would limit this potential problem.

Ergot can be an economically devastating problem. In seed production fields, yield losses ranging between 10 to 80% have been reported in India and annual losses of 10 to 25% were reported in seed production fields in Zimbabwe (Bandyopadhyay et al. 1998). Ergot is not usually a problem in grain production fields, but significant yield losses associated with ergot infection have occurred following cool mid-season conditions in some parts of Australia in 1996 and Mexico in 1998.

Although yield losses associated with ergot infection can be significant, indirect losses may be even more important. Harvesting grain from ergot infected fields can be difficult. Infected florets exude a "honey-like" substance and grain harvesting equipment can be plugged or otherwise fouled by contaminated grain. Ergot contamination reduces grain quality and limits its use as a feed-stock. Feed-refusal of ergot contaminated grain has been reported for poultry and swine. The economic impact of ergot in the United States is difficult to assess at this point. Seed production losses in Brazil were estimated at $US 3 million in 1995 (Bandyopadhyay et al. 1996). Industry reports from Australia suggest that increased management costs associated with the control of ergot in seed production fields may increase seed production costs by $20-25 per bag, a net cost of$4M per annum to the industry (Bandyopadhyay et al. 1998).

Related, Current and Previous Work

Given the recent introduction of ergot into the U.S., little is known about this disease or its potential impact on sorghum production. Two emergency research efforts were initiated in 1997 and 1998 to address this issue. NC-501, "Ergot: A new disease of U.S. sorghum production", was initiated in 1997 and included researchers from Kansas and Nebraska to address questions related to ergot biology and host-plant resistance or avoidance. NC-501 is a two-year project and will serve as the starting point for this formal NC-project proposal.

The USDA-ARS released $141 000 for projects (FY 98) working on various aspects of ergot research. Texas A&M received $84,750 for research to characterize sorghum hybrids for differences in susceptibility, to evaluate male and female fertility components of elite parent lines and determine the relationship with ergot susceptibility, and to address questions concerning ergot biology and epidemiology. Kansas State University received $35,250 to evaluate the viability of micro- and macroconidia of ergot and for research evaluating differences in mid-season cold tolerance of sorghum breeding lines and hybrids. The USDA-ARS research project in Mayaguez retained $21,000 to continue research on a dual screening technique for ergot and data collection on environmental interaction with ergot.

Most of the monetary support for ergot research to date is short-term and was obtained under an emergency mandate to address near-term disease management issues. Even though these projects were only initiated in the last year, a clearer understanding about this disease and potential control strategies is emerging. Data has been collected that indicates that ergot is capable of over-wintering in Kansas and Nebraska. Although weather conditions were not favorable for the development of ergot in grain production fields in 1998, ergot conidia collected from sorghum panicles infected in 1997 germinated in vitro and were capable of infecting sorghum panicles in the greenhouse as late as April of 1998. Chemical control strategies are being developed for seed production areas in the U.S., but these strategies have not been widely tested on farm. Methodologies for quantifying mid-season cold tolerance and ergot susceptibility are being developed. A cooperative research effort is needed to successfully confirm and implement the findings of the research that has been conducted to date.

Additional questions concerning strategies for controlling or managing ergot have yet to be addressed. The genetic and physiological basis for differences in ergot susceptibility among breeding lines and commercial hybrids have not been determined. Solutions to these questions are needed to effectively implement a disease control and management strategy.

Objectives

1. Develop a better understanding of ergot biology including epidemiology, alternate hosts, and population structure of the ergot pathogen.
   
2. Design and evaluate integrated disease control strategies for ergot including chemical and potential biological controls.
   
3. Characterize germplasm sources of host-plant resistance or avoidance to ergot and integrate these genes into elite genetic backgrounds.
   
4. 
   

Methods

1. Develop a better understanding of ergot biology including epidemiology and taxonomy of the ergot pathogen.

1.1 Epidemiology - Principle Investigators: Jim Stack, Stan Jensen, Larry Claflin, Gary Odvody.

The incidence and severity of ergot in the northern Great Plains will depend on the source of inoculum each year and the dispersal capabilities of secondary conidia. Of immediate importance is the ability of the pathogen to persist in sorghum production environments in the U.S. Will it behave similarly to rust diseases with inoculum being dispersed from the south up through the great plains each year or will it become endemic throughout the Great Plains with each region experiencing its own unique epidemic each year.

The strategy and limits of survival of Claviceps africana in the central Great Plains will be determined. To cover a range of environments (weather and edaphic factors), studies will be conducted in the seed production areas of Texas and in the grain producing areas of Kansas and Nebraska. The objectives of this research will be to quantify the potential of ergot: 1) to form sclerotia and sphacelia on sorghum tissue either on intact plants or in soil, 2) to persist in the form of sclerotia and/or sphacelia on intact plants or in soil under field and laboratory conditions, 3) to remain viable on leaf, stalk, or panicle tissues in the form of exudate produced from panicles, and 4) to survive in colonized plant tissue under exposed or sub-soil surface conditions. The impact of variable moisture, temperature, and ultraviolet radiation on survival and dispersal will be determined.

The rates of disease progress within a field, within a region, and across the central Great Plains will be determined. To assess disease progress, quantitative methods will be developed to measure disease incidence and severity. With these methods, local and regional epidemics will be characterized with respect to rate of disease development within fields and across regions. Epidemic development from Texas to Nebraska will be monitored. The influence of environmental factors on disease progress will be assessed. Attempts will be made to develop a predictive model for disease incidence and severity.

The role of potential collateral hosts (e.g., johnsongrass, shattercane, and other native range grasses) to ergot in survival will also be assessed. The source of C. africana inoculum for infection of florets during the initial flowering period in each growing region will be determined. Appropriate grass species will be grown under greenhouse conditions. At the appropriate time the plants will be induced to bloom and challenged by inoculation with C. africana spores.

1.2 Population Structure - Principle Investigators: Jim Stack, Stan Jensen

An assessment of the genetic variability within and among populations of Claviceps africana will be made by evaluating ergot isolates from Texas, Kansas, and Nebraska, as well as isolates from other samples collected across North America (e.g., Florida, Georgia). If genetic variation among C. africana populations is detected attempts will be made to determine if phenotypic variation also exists with respect to virulence and host range.

Isolates of C africana will be obtained from as many sources in the U.S. and Mexico as possible. In order to obtain quantities of macroconidia sufficient for DNA analysis, all isolates will be inoculated to susceptible sorghum under controlled conditions to ensure the establishment of pure cultures. Single spore cultures will be inoculated to susceptible sorghum to rapidly increase the biomass of the isolates and also determine the virulence of the culture. A greenhouse assay using a male sterile sorghum will be developed to permit assessment of virulence among populations of C africana. Virulence will be assessed based on infection dynamics and symptom development.

Chromosomal ITS fragments will be isolated from C. africana cultures by PCR and will be sequenced to determine species identity. At this point we are still not absolutely positive there is only one Claviceps species causing ergot of sorghum in North America. Results from our studies will be compared to results with sorghum ergot isolates in other countries (e.g. South Africa, India, and Australia). Since these foreign isolates are not available in Nebraska for analysis, comparisons will be made by collaboration with scientists in other countries or at the USDA facility in Frederick, Maryland. Mitochondrial DNA fragments will be isolated from the North American C. africana cultures and will be sequenced to determine variation among populations of C. africana. Other gene sequences already described in the genus Claviceps (reported in the literature or through interaction with colleagues) may also be used for detection of variation (perhaps using RFLP or AFLP analyses) among the C. africana populations. Should genetic variation be observed among population of C. africana, attempts will be made to correlate genetic variation with phenotypic variation (e.g., virulence).

2. Evaluate and implement integrated disease control strategies for ergot including chemical and potential biological control. 2.1 Field control of sorghum ergot - Principle Investigator: Gary Odvody

Since 1995 the systemic triazole fungicides have been effectively used to control sorghum ergot on male-sterile seed parents in hybrid seed production fields predominantly in the Western Hemisphere. Ground applications with a head-directed spray have been most effective, but triazoles differed in efficacy and some have had problems of phytotoxicity. Protection of the vast acreage of hybrid seed production fields in the U.S. will require aerial application but its efficacy is questioned due to low spray volume and poor contact and coverage of sorghum heads.

Dr, Gary Odvody from the Texas A&M Research and Extension Center in Corpus Christi, TX will coordinate and conduct field and laboratory research to evaluate chemical control strategies for ergot in hybrid seed production fields. Research objectives will include (1) characterize an array of fungicides to identify the most effective chemical agents; (2) develop timing and number of applications; and (3) identify fungicides or chemicals with differing modes of action which reduce risk of fungicide-tolerant strains of C. africana.

2.2 Control of ergot on seed - Principle Investigator: Gary Odvody

Hybrid sorghum seed harvested from ergot-affected production fields may have sphacelia/sclerotia with external and internal honeydew of C. africana and healthy seed coated with honeydew. The sphacelia/sclerotia and viable conidia in the honeydew have the potential to spread or reintroduce C africana into other sorghum growing regions. Routine seed treatments with Captan and other contact fungicides eliminate viable conidia in surface honeydew but their efficacy in preventing germination of sclerotia and conidia within sphacelia/sclerotia is unknown.

Dr Gary Odvody from the Texas A&M Research and Extension Center in Corpus Christi TX will coordinate and conduct field and laboratory research to identify a chemical or chemicals and appropriate rates which eliminate all risk of spread of C africana from ergot- contaminated seed.

2.3 Potential for Biological Management of Sorghum Ergot - Principle Investigators: Jim Stack and Gary Odvody

The honeydew exudate from infected panicles is an excellent growth medium for many microorganisms, including bacteria, filamentous fungi, and yeasts. One such colonist of ergot exudate is the fungus Cerebellum. Scientists have noted that colonization of the exudate by Cerebellum precludes the production of secondary conidia by Claviceps africana. This observation has lead to the speculation that microorganisms may be effective agents for preventing dispersal of the ergot pathogen.

The microbial composition of ergot exudate will be determined by collecting infected panicles with ergot honeydew exudation. Panicles of varying age will be collected to encompass the range from active exudation to fully weathered exudate. The exudate will be cultured for both bacteria (gram negative and gram positive) and fungi (filamentous and yeast). Isolates will be tested for their ability to prevent the production of secondary conidia by C africana on the surface of honeydew. These experiments will be conducted in the greenhouse using sorghum plants actively exuding ergot honeydew. Secondary inoculum production will be quantified and the microbial populations monitored. Since the sorghum seed is already colonized by a myriad of microbes including those found in the exudate. perhaps one source of the secondary colonizers is the seed; i.e., the honeydew is becoming colonized not just from aerial inoculum but also from the microbes in adjacent seed or in the developing flower tissues

If any of the microbial isolates exhibit the ability to prevent secondary conidium production by C africana in the greenhouse experiments, small-scale field tests will be conducted to determine whether the microbial colonists are able to prevent the production and/or the dissemination of secondary conidia by Claviceps africana under field conditions.

3. Characterize germplasm sources of mid-season cold tolerance and ergot disease resistance and integrate these genes into elite genetic backgrounds.

3.1. Pollen management - Principle Investigators: Mitch Tuinstra, Ken Kofoid, Jeff Pedersen

Cool wet weather during flowering can reduce male fertility and lead to conditions that favor ergot infection in grain production fields. This opportunity for ergot infection can be greatly reduced by utilizing hybrids that maintain pollen production and viability. Mid-season cold tolerance as a mechanism of ergot avoidance is likely to be an important component of ergot disease control in the cooler sorghum producing environments of the U.S. including the high-plains of Kansas and Nebraska.

Genetic variability for mid-season cold tolerance exists in the sorghum gene pool (Brooking, 1979). A preliminary analysis of mid-season cold stress responses of sorghum at Kansas State University and the University of Nebraska has indicated that cold temperature stress primarily influences the timing of anther dehiscence and consequently pollen production. An analysis of elite parent lines under controlled environment conditions has indicated that some lines shed pollen normally following a 12 h cold stress treatment but anther dehiscence is delayed in others.

An experiment will be conducted by sorghum breeders in Kansas and Nebraska to evaluate mid-season cold tolerance and ergot susceptibility in elite commercial hybrids and breeding lines. Breeding lines and commercial hybrids will be evaluated for differences in cold tolerance under controlled environment conditions. Differences in cold tolerance will be quantified by excising panicles or panicle branches from field grown plants and subjecting these panicles to a cold temperature treatment for 12 h. Pollen production, timing of anther dehiscence and pollen germination will be determined at 30 min intervals following the cold treatment. The results of these experiments will be pooled across locations to determine repeatability and consistency of this response. A replicated field study at high-altitude nursery locations in Mexico will also be conducted to evaluate these breeding lines and hybrids for differences in pollen production and ergot susceptibility under natural field conditions. Differences in timing of anther dehiscence, quantity of pollen produced, and ergot susceptibility will be measured. Data will be pooled across locations to determine the relationship between field and laboratory measurements.

3.2 Host plant resistance

3.2.1. Line x Tester Analysis - Principle Investigators: Mitch Tuinstra, Ken Kofoid, Jeff Pedersen

Considerable variability in ergot susceptibility has been reported among sorghum lines and hybrids. Recent studies of host-plant resistance to ergot have focused on the analysis of testcross hybrids in cytoplasmic male-sterile backgrounds (A3). Hybrids developed in A3 cytoplasm are male-sterile so mechanisms of ergot escape are circumvented and the results of germplasm screening experiments are more easily interpreted. In one such study conducted by USDA-ARS researchers in Puerto Rico, the sorghum accession IS 8525 expressed low levels of disease incidence and severity in both male-fertile and male-sterile genetic backgrounds. The expression of host-plant resistance from this source needs to be further characterized to determine it's potential for use in breeding.

Sorghum breeders in Kansas and Nebraska will conduct replicated experiments to evaluate stigma receptivity, seed production, and ergot resistance characteristics of IS 8525 in a line x tester analysis. IS 8525 and standard seed parent lines will be crossed to A3TX7000, A3TX2737 and ASKS70. The resulting testcrosses represent a series of male-sterile hybrids that can be used to evaluate the mode of inheritance and combining ability effects of these parent lines on host-plant resistance to ergot and on seed setting characteristics. The results of this study should also reveal any negative seed production characteristics associated with the resistance trait and will indicate the feasibility of introducing host plant resistance into seed parent lines and hybrids.

Testcross hybrids will be synthesized during the summer of 1999. Lines and testcross hybrids will be evaluated for ergot susceptibility in replicated trials at winter nursery locations in Puerto Rico and Mexico. Replicated trials to evaluate pollen receptivity and seed setting characteristics of the parent lines and testcross hybrids will be conducted at three locations in Kansas and Nebraska. Controlled pollinations will be used to quantify the seed setting efficiency of sterile hybrids. The results of experiments will be pooled to evaluate line and tester effects on these traits and the relationship between ergot resistance and seed production characteristics.

3.2.2 Breeding for host-plant resistance - Principle Investigators. Mitch Tuinstra, Ken Kofbid, Jeff Pedersen

IS 8525 is poorly adapted for seed production. Host-plant resistance traits must be transferred to elite U.S. seed parent lines before they will be considered for use by the sorghum seed industry. IS 8525 is being introgressed into several elite U S seed parent backgrounds to develop populations for breeding and genetic research. First and second generation backcross populations are being developed in Kansas and will be evaluated for ergot resistance as they become available.

Measurement of Progress and Results

Outputs

Outcomes or Projected Impacts

• This research will impact all levels of the sorghum seed and grain production industry. Given the general lack of information on ergot of sorghum, each of the experiments described in this proposal will contribute new information to our understanding of ergot control in sorghum.</P> <P>The main focus of the first research objective is on ergot biology and disease control. Developing a clear understanding of the ergot life cycle will be crucial in developing new ergot control strategies. The development of effective chemical control strategies will play a crucial role in the sorghum breeding and seed production industries in the U.S. This is an immediate and important need of the sorghum seed industry and should serve to stabilize and ensure adequate seed production.</P> <P>The sorghum genetic research outlined in this proposal should contribute to improved screening strategies and knowledge concerning mechanisms of host-plant resistance or disease avoidance. The development of reliable screening strategies for mid-season cold tolerance should enhance the efficiency of breeding for cold tolerant sorghum parent lines and will provide a uniform test for comparing cold tolerance among commercial hybrids. Breeding for host-plant resistance to ergot is an entirely new area of research. The experiments outlined in this proposal will provide basic information to determine the feasibility of host-plant resistance to control ergot in seed and grain production fields. The success of this program will contribute to reduced potential for ergot infection of seed production fields, thereby reducing seed costs. Host-plant resistance will also reduce the potential for sporadic ergot infection epidemics in grain production fields.</P> <P>The results of this research will be communicated at scientific meetings, through abstracts and proceedings, and in refereed journal publications. These results should have direct application in the sorghum seed industry and should indicate new and potentially useful directors for research.

Milestones

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Projected Participation

View Appendix E: Participation

Outreach Plan

Organization/Governance

The technical committee will meet once annually with the administrative advisor to review research progress and to plan future research. These meetings will rotate among participating North Central and southern U.S. Agricultural Experiment Stations and USDA-ARS research units to spread out travel costs and provide exposure to various research programs. An annual report will be submitted to the administrative advisor and Technical Committee.

Officers of the proposed committee will be a chair and secretary. The secretary, elected during the annual meeting will succeed the chair at the time the annual report is filed with the administrative advisor. The responsibility of the committee chair will be to organize committee meetings, appoint subcommittees when necessary, and prepare the annual report. The secretary will record minutes of meetings and will act as vice-chair.

An Executive Committee will consist of the chair, secretary, immediate past chair, and administrative advisor. The Executive Committee will consider any administrative issues arising between annual meetings of the entire committee.

Given the multidisciplinary nature of research focused on ergot control, contacts with other plant pathologists, physiologists, breeders, etc. will be encouraged. Industry representatives from the sorghum seed industry will also be encouraged to contribute research ideas and attend meetings.

Literature Cited

Arias-Rivas, B., Silva, F., and Cabrera, N. (1997). Sugary disease or ergot in sorghum seed fields on the Guanipa Plateau, Anzoategui State, Venezuela. International Sorghum and Millets Newsletter 38, 64-67.

Ayyangar, G.N.R., and Rao, V.P. (1931). Studies in sorghum: I. Anthesis and pollination. Indian Journal of Agricultural Science 6, 1299-1322.

Bandyopadhyay, R., Frederickson, D.E., McLaren, N.W., and Odvody, G.N. (1996) Ergot - a global threat to sorghum. International Sorghum and Millets Newsletter 37, 1-32.

Bandyopadhyay, R., Frederickson, D.E., McLaren, N.W., Odvody, G.N., Ryley, M.J. (1998). Ergot: A new disease threat to sorghum in the Americas and Australia. Plant Disease 82, 356-367.

Bennett, W.F., Tucker B.B., and Maunder, A.B. (1990). Modern Grain Sorghum Production. Iowa State University Press, Ames, Iowa.

Brooking, I.R. (1979). Male sterility in Sorghum bicolor induced by low night temperature, II. Genotypic differences in sensitivity. Australian Journal of Plant Physiology 6, 143-147.

Frederickson, D.E., and Mantle, P.G. (1988). The path of infection of sorghum by Claviceps sorghi. Physiological and Molecular Plant Pathology 33, 221-234.

Frederickson, D.E., Mantle, P.G., and De Milliano W.A.J. (1991). Claviceps africana sp. nov.; the distinctive ergot pathogen of sorghum in Africa. Mycological Research 95, 1101-1107.

McLaren, N.W., and Wehner, F.C. (1992). Pre-flowering low temperature predisposition of sorghum to sugary disease (Claviceps africana). Journal of Phytopathology 135, 328-334.

Attachments

Land Grant Participating States/Institutions

IN, KS, NE, TX, WI

Non Land Grant Participating States/Institutions