Plant Diseases National Program

National Program Overview:  ARS Research is organized into 22 National Programs. These programs serve to bring coordination, communication and empowerment to the more than 1200 research projects carried out by ARS. The National Programs focus on the relevance, impact, and quality of ARS research. These National Programs are organized within four broad areas:  Nutrition, Food Safety/Quality (NFSQ); Animal Production and Protection (APP); Natural Resources and Sustainable Agricultural Systems (NRSAS); and Crop Production and Protection (CPP).  The Plant Diseases National Program (NP303) is part of CPP but interaction also occurs with researchers in APP, NRSAS and NFSQ.  The annual budget is $66,593,891 and 158 scientists conduct research at 41 locations.

Each National Program conducts a planning workshop to focus the research program by learning the problems and needs of customers, stakeholders and partners.  These workshops help ensure that our research programs are relevant to the concerns of our constituents. The Plant Diseases National Program Workshop was held in Beltsville, Maryland, October 1999.  Approximately 70 customers and stakeholders and 50 ARS scientists attended.  The participants identified the important disease problems facing their crops and commodities.  Ongoing research in each problem area was discussed.

Based on input received at the 1999 planning workshop, research in the Plant Diseases National Program was organized into 5 component areas:  Identification and Classification of Pathogens; Biological Control; Cultural Control; Pathogen Biology, Genetic, Population Dynamic, Spread and Relationship with Hosts and Vectors; and Host Plant Resistance to Disease.  Teams of ARS Scientists worked with National Program Leaders to develop an Action Plan that provided the framework for ARS research over a five-year period.  Each of the five components contained problem areas that required research.  Groups of ARS scientists wrote Project Plans that described the research they would conduct, the anticipated products or information to be generated by the research, roles and responsibilities of ARS scientists and their cooperators, and timelines and milestones to measure progress toward achieving the goals of the research.  These project plans were reviewed for scientific quality by a panel of experts in the field.  ARS used input from the panel to revise and improve their planned research.

The final stage in the cycle of an ARS National Program involves assessment of research that has been conducted.  This expert panel on which you are a member, is assembled to evaluate the impact of ARS research accomplished and to offer suggestions for future ARS research direction and emphasis.  Input from this panel and from interested parties at a planning workshop to be held May 17-19, 2005, in Orlando, Florida, will be used to develop the framework for ARS research over the next five years.

Program Summary:  The overall goal of the Plant Diseases National Program is to develop and improve ways to reduce crop losses caused by plant diseases.  The program focuses on developing effective disease control strategies that are not environmentally harmful, do not threaten the safety of consumers, and are compatible with sustainable and profitable crop production.  The ARS program is conducted in cooperation with related research in other public and private institutions.

Plant diseases, caused by viruses, viroids, bacteria, phytoplasmas, fungi, and nematodes, result in economic losses in agriculture, landscape, and forest settings by reducing yields, lowering product quality or shelf-life, decreasing aesthetic or nutritional value, and, sometimes, by contaminating food and feed with toxic compounds.  Control of plant diseases is essential to providing an adequate supply of food, feed, and fiber.  Growers currently spend large sums to achieve partial control of pathogens that attack crops and other plants.  Even then, crop and commodity losses caused by diseases cost billions of dollars each year.  Reducing such losses has long been a high priority objective for agriculture and also for the Agricultural Research Service (ARS).  Besides the obvious monetary benefits for producers and processors, successful plant health protection is important if we are to maintain and increase food supplies with minimum increases in land under cultivation.  Additionally, knowledge and management of plant diseases of quarantine significance are vital, not only for protecting our domestic crops from foreign disease, but also for maintaining and expanding export markets for plants and plant products. 

Strategies for the control of plant diseases include:  planting resistant crop varieties; changing crop cultural practices or storage conditions to those less favorable for disease development; employing biological controls; applying chemical pesticides; and using integrated disease management (combining two or more of the above approaches).  The ability to develop any of these strategies depends first on identifying the pathogen causing the disease, then learning how to interrupt its disease cycle.  The more known about the genetic, biochemical, and physiological processes that operate in the host and pathogen as infection and disease progress, the more likely a control method can be devised.  Understanding the ecology of pathogens (that is, how they survive, are dispersed, and otherwise interact with their environment) is also important.  In addition, an understanding of the epidemiology or outbreak of disease and spread of pathogens is important for deciding which control actions are most effective.

As with all microorganisms, plant pathogens exhibit a remarkable ability to change and adapt.  Newly discovered pathogens and more virulent strains of old pathogens continually arise and often overcome resistant crop varieties or can no longer be controlled by strategies and chemicals that were once effective.  Continual research to develop new control methods is necessary to increase or even maintain current levels of crop production and commodity preservation.  Further complicating the situation, public concern has grown in recent years regarding the use of synthetic chemicals to control diseases.  This concern stems from the fear that such chemicals may contaminate food or accumulate in the soil and ground water (and so be introduced into the food chain).  As a result, pressure has increased to develop nonchemical approaches to plant disease control.  Typically, nonchemical controls do not exist, are less effective, or too costly.  More research is needed to develop or improve nonchemical control methods and procedures. 

Host plant resistance to plant diseases is probably the most desirable control strategy because it can be highly effective, is environmentally benign, and usually entails little or no additional expense to producers.  To be most effective, such resistance should be durable, i.e., not readily overcome by mutations in pathogens that increase their virulence.  This requires detailed knowledge of the pathogens that are present, their ability to change, and the nature of the resistance processes themselves.

For diseases where host plant resistance is unavailable or only somewhat effective, strategies are needed that integrate cultural, biological, and chemical control procedures.  For example, different crop rotations or improved cultural practices can sometimes suppress development of pathogenic organisms while maintaining a high level of productivity.  Biological control shows great potential for disease control, but has often been unreliable or only marginally effective.  Additional research is needed to understand the interactions of microorganisms in the soil, plant parts, and on the mechanisms that biological control agents use to reduce or prevent the onset and development of disease.  Finally, the appropriate use of chemicals will continue to be an important tool in the battle against plant diseases, especially where alternative controls are not effective.  Additional research is needed to develop safer chemicals and to increase the efficiency of chemical applications.  

Also required are rapid, reliable pathogen detection and identification procedures for accurate and timely disease diagnoses.  Improved detection and identification procedures are also becoming more important as international trade of plant products increases and as trading partners seek to protect themselves from the introduction of unwanted diseases. 

Recently developed biotechnology tools provide promising new approaches for achieving control of diseases.  Genetically engineering plants for resistance to viral, bacterial, fungal, and nematode pathogens has shown some success, but more research is needed.  Also, techniques have been developed that allow researchers to track specific resistance genes, facilitating breeding resistant varieties.  Biotechnology offers the exciting possibility of developing disease resistance in plants that cannot be accomplished through conventional breeding procedures (for example, by introducing genes for resistance from unrelated species). 

Additionally, Congress has appropriated funds to help solve specific production and marketing problems of the United States industry.  ARS has decided that some of the funds will be used for agreements with State Agricultural Experiment Stations and similar research institutions.  Proposals are solicited by the National Program Staff and are evaluated by ARS, state scientists and industry representatives.  Criteria used to evaluate the proposals include: national priority of the research, scientific expertise of those involved in the project, funding requested, and probability of success in solving production and marketing problems.    

Funds are maintained at ARS headquarters because the program covers several regions and national programs.  Proposals funded are designed to demonstrate accomplishments in key areas aligned to the objectives of the Plant Diseases National Program:  key diagnostic tools for identification of pathogens, resistance genes to devastating diseases, understanding of pathogen biology, and epidemiology and vector relationships.  Current projects funded by this process include:  The Potato Research Program, The Small Fruit and Nursery Research Program, The Fusarium Head Blight Program, and The Floral and Nursery Research Initiative. 

Identification and Classification of Pathogens Component

Background:  Effective disease control depends on rapid and accurate identification of the pathogens involved so that appropriate control measures may be taken.  Accurate identification of pathogens is also critical for making sound decisions regarding quarantines of imported and exported plant materials and commodities.  Knowing how pathogens are related to each other can be helpful in suggesting possible control strategies.  ARS research has significantly impacted science and the public because of accomplishments in identifying, characterizing, and classifying a broad spectrum of pathogens.

Structural and functional genomics of potato, strawberry, grapevine, jujube, walnut, bean, carrot, peach, cherry, pear, and almond pathogens:  Plant diseases caused by vascular system-inhabiting wall-less bacteria (mollicutes, phytoplasmas and spiroplasmas) and walled bacteria are responsible for significant economic losses to U.S. and world agriculture. Accurate, rapid and sensitive technologies for detection and identification of these pathogens and their ecologies are vital prerequisites for decisions to formulate appropriate disease control measures and for intercepting pathogens under quarantine regulations. Knowledge gained from structural and functional genomics is making it possible to devise improved and novel means for genus-level, species-level, and virulent strain-level detection and identification of these pathogens, including invasive species. Comparative genomic studies will lead to identification of potential genes useful for detection and identification of the pathogens at various taxonomic levels. Genomic information and the new knowledge of bacterial pathogenesis will promote fundamental and applied research on disease control.

Floral and woody ornamental plant prokaryotes:  Bacterial diseases caused by Pseudomonas, Xanthomonas, Xylella, Ralstonia and other genera often result in significant losses in the production and quality of ornamental crops, and are very difficult to control. Although X. fastidiosa has been known to cause leaf scorch diseases of woody ornamentals for 20 years, some basic questions still remain largely unanswered. These include the host range of X. fastidiosa in horticultural and alternative plants, and the genetic and pathogenic relationships among strains of X. fastidiosa isolated from economically important horticultural and agricultural hosts and from invasive alternate hosts in the environment. Recent surveys indicate that the disease is spreading and becoming more severe in important landscape trees such as oak, elm and sycamore in many states. Bacterial leaf scorch of oleander, a relatively new disease reported in 1999, is also an emerging problem in California, Arizona and Texas where oleander is used as a popular landscape plant for residential hedges, highway dividers, colorful accents and beach plantings. In order to effectively control bacterial leaf scorch diseases of landscape trees and shrubs, answers to the above questions are greatly needed.

Crown gall and deep bark canker (DBC) of nut trees (walnuts/almonds:  Crown gall, caused by the bacterium Agrobacterium tumefaciens, is the most important disease of walnuts in California; there is no known effective control strategy. The disease is present in all walnut-growing regions of the state and can occur on 80% of the trees in a given orchard causing significant yield losses, and sometimes tree death. Initial efforts are focusing on the design and development of robust detection methods for Agrobacterium. In the southern range of the walnut-growing region, DBC (Brennaria rubrafaciens) is eliminating the use of a walnut cultivar that once represented over 60% of the walnut industry.  As with crown gall, there are no effective control measures for DBC. Consequently, ARS scientists are designing and developing robust detection methods for Brennaria.

Exotic prokaryotic pathogens of citrus, cereals, watermelon, grape, shade trees, potato tomato, grass seed, and rice:  Foreign species of plant-pathogenic bacteria have become a major threat to U.S. agriculture because of the increase in air travel and international agricultural trade. The accidental or deliberate release of a regulated bacterial plant pathogen could result in severe economic damage. Researchers used an unpublished, newly developed pathogen-rating software program to determine the threat of naturally or deliberately released pathogens. The researchers then selected four high-threat foreign bacteria:  Rathayibacter species, Xylella fastidiosa, Xanthomonas campestris pv. citri, and Xylophilus ampelinus.

Molecular and Morphological Systematics of Plant-Pathogenic Fungi:  Economic loss to agricultural and horticultural crops due to disease-causing fungi is estimated at $20 billion per year in the United States.  In addition, fungi often limit U.S. agricultural exports, a significant factor in the U.S. balance of trade.  Many of these pathogens have several morphologically similar relatives with no quarantine significance. Rapid and accurate methods for identification of quarantine significant fungi are critical to protect U.S. export markets.  The $6 billion wheat export market was threatened by the presence of Karnal bunt and morphologically similar fungi, including a bunt fungus on ryegrass found throughout the southeastern United States.  Other beneficiaries of similar research include the soybean, fruit, and tree nut industries.

Phytophthora spp. of strawberry, nut trees (walnuts/almonds), stone fruit and grape:  This project focuses on determining the etiology of and improved management strategies for key diseases of small fruits and deciduous tree crops includes the development of improved diagnostic detection methods for pathogenic Phytophthora spp. affecting strawberry and deciduous fruit and nut crops.  PCR primers are being used for specific amplification of mitochondrial DNA from P. cactorum, P. citricola, and P. megasperma extracted from soil and plant samples. 

Alternaria diseases of fruit trees:  Comprehensive knowledge of the biology of microorganisms associated with tree fruits, including pathogen taxonomy, disease etiology, and disease detection is lacking. Such knowledge is critical for development of science-based phytosanitary regulations and mitigative measures for imported and exported tree fruits. Mis-identification of fungal and bacterial pathogens of tree fruits can lead to the importation of exotic pathogens and diseases, and can hinder progress in development of export markets for U.S. commodities. To address these issues ARS scientists are studying pathogen biology of selected agents, including Alternaria spp. (causing several different fruit diseases) and are conducting morphotaxonomic and DNA fingerprinting studies necessary to develop DNA-based methods for detecting and identifying exotic and domestic fruit pathogens in this genus.

Fungal diseases of cotton:  Plant pathogens and nematodes cause annual yield losses of between 11 and 13%, accounting for over $800 million in losses to U.S. cotton growers per year.  Despite the introduction of new elite varieties, cotton yields have been stagnant for over a decade. The causes of yield stagnation are varied and complex, but the emergence of new pathogens, the spread of recognized pathogens, and the tenacious resiliency of established pathogens are significant factors.  Specific pathogens or diseases include the reniform nematode, race 4 of Fusarium oxysporum f. sp. vasinfectum, bronze wilt, South Carolina seed rot, and seedling diseases caused by Rhizopus oryzae and other pathogens. The multi-prong attack strategy required to combat these problems includes the development of resistant varieties, the identification of pathogenic organisms, the development of effective biocontrol agents, and the acquisition of a knowledge base encompassing both the mechanisms of pathogenicity and the factors affecting plant resistance.

Soybean rust:  Soybean rust is an important disease causing significant yield losses in most soybean growing regions throughout the world, except for the United States. At least two species of Phakopsora cause soybean rust. Asian isolates of the soybean rust fungus Phakopsora pachyrhizi have been reported to be more virulent and aggressive than those of P. meibomiae from South and Central America and Puerto Rico. The Asian species of soybean rust has been estimated to result in yield losses of over 10% if it becomes established in the continental U.S. In November 2004, soybean rust was found in the United States for the first time, and since then has spread to 9 states.

Soilborne fungal pathogens of alfalfa and dry beans:  Alfalfa is the fourth most important crop in the United States in terms of both cultivated acreage and production value. Approximately 25% of the alfalfa hay crop of the United States is lost annually to disease, principally the result of soilborne pathogens. Conventional chemical approaches to disease control are too expensive, ineffective, or deleterious towards non-target organisms and the environment. The most effective methods for minimizing losses from disease are to avoid infested fields and to grow alfalfa cultivars that are resistant to multiple diseases. Timely detection of pathogens in fields using microbiological methods is impeded by an unavailability of selective media for specific pathogens, or the long incubation periods required for unambiguous pathogen identification. PCR-based assays are needed to accurately and quickly detect soilborne pathogens of alfalfa in infected plants and infested field soil. Real-time PCR assays that quickly and reliably quantify pathogen biomass in infected tissues would facilitate the accurate selection of highly resistant plants.

Sudden oak death and the ornamentals industry:  Sudden oak death (SOD) is caused by Phytophthora ramorum, a newly described species found in 1993 to attack ornamentals in Germany and the Netherlands. The same pathogen (as determined by morphology and isozyme analysis) has been known since the mid-1990's to be responsible for death of thousands of oaks in California.  Sudden oak death has become of major concern to the ornamentals industry and other agricultural industries because of state, federal, and international quarantines (Canada) affecting the movement of susceptible, high-value nursery crops such as rhododendrons and azaleas, as well as firewood and other plant-related products. The list of hosts of P. ramorum is now over 30 in number and growing, but little is known about the susceptibility of potential host species in the Eastern United States. Because several west coast nurseries unknowingly shipped thousands of infected plants throughout the United States in 2003 and 2004, major efforts are underway to prevent P. ramorum from becoming established in the East.

Molecular and Morphological Systematics of Plant-Pathogenic Nematodes:  Plant-parasitic nematodes are microscopic soil worms that reduce the yield and quality of many crops, with annual agricultural losses of over $10 billion in the United States. Accurate and rapid identifications are urgently needed to determine the extent of nematode damage and to develop nematode control strategies. Because identification can be a difficult and time-consuming process, ARS scientists are improving the science of nematode identification through compiling guides or "keys" for identifying nematodes, updating lists of crops damaged by specific nematodes, maintaining nematode collections, determining new traits useful for identifying nematodes, and placing them in family trees in order to predict their agricultural effects. In addition, some beneficial nematodes from soil and insects are being similarly characterized. Identifications of deterious nematodes from plant materials introduced to or exported from the United States limit the international and interstate spread of nematodes and permit export of billions of dollars of agricultural products monitored by regulatory agencies. Although fruit and nut trees, ornamentals, potatoes, soybean, vegetables, and wheat are the major crops impacted by ARS nematode systematics activities, the scope of this project is truly multicommodity.

Soybean cyst nematode:  Each year, the quantity and quality of soybean produced in the United States are adversely affected by a wide range of pathogens and pests, which cause a billion dollar crop loss annually. Disease-causing pathogens are being characterized in order to produce new soybean lines that are resistant to diseases. Investigations are being done to identify host genes that permit the vertical transmission of viruses and virus genes that reduce seed quality. These investigations will reduce the impact of diseases on soybean production by enhancing soybean germplasm with resistance to pathogens, by creating knowledge of the host-pathogen interactions that lead to disease, and by developing sustainable agricultural practices.

Exotic viruses of stone fruits and citrus:  Exotic viruses of stone fruits and citrus and their potential insect vectors pose threats to our agricultural system through natural or intentional introductions. The protection of our agricultural production systems depends on the development of detection technologies, identification of causal agents and potential mechanisms of transmission, and determination of factors required for disease management, eradication, or prevention. Because viruses readily mutate and genetically recombine, understanding the dynamics of population change improves the probability of control, prevention, and eradication.

Viruses of citrus, vegetables, ornamentals and stone fruits:  Multiple pathogens are responsible for production losses in the U.S. citrus, vegetable, ornamental and stone fruit industries. New and/or more severe plant diseases may indicate the existence of new plant pathogens or the appearance of more virulent strains of existing pathogens. In both cases, the synthesis of useful tools that diagnose and differentiate pathogens requires the integration of data on the biological, serological and genetic attributes of these pathogens. The successful management of existing disease problems can benefit from new diagnostic tools developed with data generated from the application of new genetic technologies to pathogen identification and differentiation not possible using previous research capabilities.

Viruses of floral and woody ornamental plants:  Many plant virus diseases cause significant losses in the production and quality of ornamental crops. These diseases are very difficult to control, and new diseases occur as different crops are introduced or grown in new areas. Many crops are susceptible to multiple viruses, each of which may cause serious economic losses, and infected plant material may not be acceptable for sale or export. Methods to reliably and rapidly detect and identify these viral pathogens are necessary for the production of virus-free or virus-indexed plants. One primary focus of ARS research is on those "new" currently uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry. An understanding of the identity of new and emerging ornamental viruses, as well as their mechanisms of infection, transmission, and pathogenicity, is needed in order to develop better methods of disease control. 

Exotic viruses of sweet potatoes:  Federal regulations prohibit the entry of sweet potato germplasm from foreign sources for propagative purposes. Small quantities of germplasm are allowed for scientific uses, if the material undergoes quarantine and indexing for exotic pathogens (primarily viruses and phytoplasmas). ARS is responsible for conducting the quarantine and testing of sweet potato germplasm. Sweet potato virus disease (SPVD) is among the most serious diseases of sweet potato worldwide. Previously, it was thought to be caused by a synergistic interaction of two viruses, sweet potato feathery mottle virus (SPFMV, a potyvirus) and sweet potato chlorotic stunt virus  (SPCSV, a crinivirus). However, the etiology of the disease is poorly understood and considerable variation has been reported in the symptoms and severity. In order to find effective control, elimination, or prevention methods for the disease, a better understanding of the disease etiology is needed.

Grapevine rootstock stem lesion complex and young vine decline of grape:  In grapevines, young vine decline (YVD) was reported in the early 1990’s and was found to be associated with several fungal pathogens. However, ARS researchers have discovered a new virus strain and several new graft-transmissible agents (GTAs) that have significant impacts on vine health and productivity and are associated with YVD. Researchers are also discovering and characterizing new GTAs that are involved in another problem in grape production, grapevine rootstock stem lesion.

Viruses of strawberry, blueberry, blackberry and raspberry:  The yield and quality of small fruit crops in the United States are adversely affected by viruses transmitted by soilborne and aerial vectors. Viruses have been implicated in decline symptoms in blackberry production fields in the southern and southeastern United States, black raspberry fields in the northwest and north central United States, and strawberry fields in California, Oregon, Washington and the Northeast, as well as in the newly described “fruit drop” and “no blossom” diseases in blueberry. In each of these crops, the available laboratory tests for viruses failed to detect any virus associated with the disease symptoms. Lack of adequate knowledge about the etiology and epidemiology of these diseases precludes development of environmentally sound, effective and economic disease control management strategies. There is a need to develop rapid assay procedures for the uncharacterized viruses in the small fruit crops so that the health status of plants can be determined in a timely manner.

Rhizomania of sugarbeet and other viruses of sugarbeet, lettuce, tomato, cucurbits, and

Strawberry:  Several economically significant soilborne and insect-transmitted virus diseases affect the sugarbeet and vegetable industries of California, the rest of the United States, and the world. Previously unknown viruses continuously emerge and cause significant economic losses, while older viruses re-emerge to once again impact production. The emergence of new viruses has been exacerbated in recent years by the international movement of plant materials and their vectors, increased farming, farming in new areas, and global climatic changes. ARS research is directed at detection and identification of new viruses as they emerge in affected crops. Specific biological and molecular approaches are being used to elucidate the etiology and molecular evolution of sugarbeet, vegetable and interrelated crop and weed viruses.

Multidisciplinary: The Electron Microscopy Unit:  A wide range of commodities  are impacted by the research of the EMU at the Beltsville Agricultural Research Center (BARC), including soybean, potato, vegetables, grain crops, cacao, coffee, citrus, and weed species. The mission of the EMU is (i) the application of an array of electron microscopy techniques to the research needs of scientists at Beltsville and other locations and (ii) the development of new techniques and methodologies in electron microscopy for achieving previously unobtainable data. Standard scanning electron microscopic observation is carried out after solvent dehydration, critical point drying and heavy metal sputter coating. This procedure often yields specimen materials that are collapsed, detached from their host materials and cleansed of any soluble surface materials making accurate taxonomic description and host/pathogen relationships difficult to accurately observe. Consequently, the EMU is developing improved methods of specimen preparation and fixation. Although this work spans all of the projects studied at BARC, it does focus on plant pathogens and pests.

Discovery Area 1:  Identification of Pathogens New to Science or Unknown in the United States

Fungi

Alternaria diseases of fruit trees

Accomplishment: At the request of APHIS-PPQ, from 2001-2003 an ARS scientist examined fruit from multiple shipments of Ya Li pears from China that were intercepted at a U.S. port-of-entry because of extensive decay caused by Alternaria. Isolations of Alternaria were made from the fruit, and pathogenicity testing was completed with representative Chinese strains and with other fruit-associated Alternaria species. After morphological and genetic characterization of the Chinese strains, the causal agent was determined not to be A. gaisen nor A. alternata (as claimed by the Chinese government) but instead was at least two undescribed species unknown in the United States. One of these was recently described as the new species A. yaliinficiens. This species is now known as one of the etiologic agents of the disease now referred to as chocolate spot of Ya Li pear.

Impact: This work has protected the U.S. pear grower from introduction of potentially damaging exotic fruit pathogens. The Chinese Ya Li pear import program has been opened and then suspended every year since 2001 because of repeated interception of the exotic Alternaria spp. On December 15, 2003, an ARS scientist discovered in a Wenatchee, WA retail outlet Chinese Ya Li pear fruit that had passed the import inspection procedure but possessed symptoms of chocolate spot disease. On December 19, 2003 APHIS issued a nationwide recall notice for all Ya Li pear fruit of Chinese origin. Within 24 hours of the notification to APHIS, the presence of diseased Ya Li pears was confirmed in 18 cities across the United States. APHIS smuggling interdiction teams were dispatched across the United States; the teams seized, destroyed, or allowed re-export of more than 3.25 million pounds of imported Chinese Ya Li pears in the United States. Diseased fruit was detected in 666 retail outlets. More than 60 sea containers of Ya Li fruit en route from China were refused entry into the United States, and this import program remains closed today, pending new information on the biology of the pathogens and development of effective mitigative measures.

Documentation:

Roberts, R.G., Andersen, B.A., Reymond, S.T. RAPD fragment pattern analysis and morphological segregation of small-spored Alternaria species and species-groups. Mycological Research. 2000. V. 104(2). P. 151-160.

Andersen, B.A., Kroger, E. Roberts, R.G. Chemical and morphological segregation of Alternaria arborescens, A. infectoria and A. tenuissima species-groups. Mycological Research. 2002. V.106(2). P. 170-182.

Roberts, R.G. Evaluation of buffer risk associated with fire blight and export of mature apple fruit. Acta Horticulturae. 2002. V. 590. P. 47-53.

Berbee, M.L., Payne, B.P., Zhang, G., Roberts, R.G., Turgeon, G. Shared ITS DNA substitutions in isolates of opposite mating type reveal a recombining history for three presumed asexual species in the filamentous Ascomycete genus Alternaria. Mycological Research. 2003. V. 107(2). P. 169-182.

Roberts, R.G. Alternaria Yaliinficiens sp. Nov. on Ya Li pear fruit: from interception to identification. Plant Disease. 2005. V. 89. P. 134-145.

Fungal diseases of cotton

Accomplishment: ARS scientists conclusively established that two species of Pantoea are etiological agents that cause South Carolina Seed Rot. Rhizopus oryzae was identified as a virulent soil-borne pathogen inciting pre-emergence damping-off in susceptible cotton cultivars. A unique and highly virulent strain of Fusarium oxysporum f. sp. vasinfectum was discovered in cottonseed imported from Australia into California as a feed for dairy cows. This strain has devastated cotton production in areas of Australia where it occurs, resulting in 98% yield losses.

Impact:  Identification of the causal agent of South Carolina Seed Rot is the first step required to develop strategies to control this disease. Identification of the highly virulent isolate of Fusarium oxysporum f. sp. vasinfectum from Australia resulted in the initiation of steps to more thoroughly disinfest cottonseed before it is allowed into the U.S.

Documentation:

Howell, C.R. 2002. Cotton seedling pre-emergence damping-off incited by Rhizopus oryzae and Pythium spp. and its biological control with Trichoderma spp. Phytopathology. 92:177-180.

Bell, A.A., Wheeler, M.H., Liu, J., Stipanovic, R.D., Puckhaber, L.S., Orta, H. 2003.Studies on polyketide toxins of Fusarium oxysporum f. sp. vasinfectum: Potential targets for disease control. Pest Management Science. 59:736-747.

Medrano, E.G., Jones, M., Bell, A.A. 2004. Association of Pantoea agglomerans with seed rot of South Carolina cotton. Phytopathology. 94:S69.

Nematodes

Molecular and Morphological Systematics of Plant-Pathogenic Nematodes

Accomplishment:  Since 2000, ARS has provided taxonomic expertise for 2,724 nematode samples received from APHIS personnel (406 urgent samples and 682 non-urgent) and federal, state and foreign scientists for research, regulatory and control purposes. In response to a request from APHIS for species identification, ARS examined the ribosomal intergenic spacer (IGS) and mitochondrial sequences of a suspicious root-knot nematode from Florida and confirmed that it was Meloidogyne mayaguensis, a resistance-breaking tropical species not previously found in the continental United States. Descriptions of new species of lesion nematode, cyst nematode, needle nematode and two root-knot nematodes incorporating morphological, molecular and host characterizations were made, thereby identifying the causal agents of newly discovered crop losses.

Impact: Identifications for APHIS personnel allowed port-of-entry personnel to prevent the spread of dangerous nematodes and also aiding the export of millions of dollars of U.S. agricultural products. Confirmation of the identity of M. mayaguensis resulted in increased sampling for this resistance-breaking nematode; multiple detections in Florida have resulted in changes in agronomic practice. Morphological plus molecular descriptions for new species led to control recommendations for new diseases and increased the reliability of species diagnoses by all nematode diagnosticians.

Additional Information: Collaboration with APHIS has been a major source of live material and identification requests behind these accomplishments.  Several university researchers have also informally contributed materials and expertise.  APHIS provides funding each year to partially defray the costs of providing identifications.

Documentation:

Handoo, Z. A., Nyczepir, A.P.,  Esmenjaud, D., van der Beek, J.G.,  Castagnone-Sereno, P.,  Carta, L.K.,  Skantar, A.M., Higgins, J.A. 2004. Morphological and molecular characterization of Meloidogyne floridensis n. sp. (Nematoda: Meloidogynidae), a root-knot nematode parasitizing peach in Florida. Journal of Nematology. V. 36 p.20-35.

Handoo, Z.A., Carta, L.K., Skantar, A.M. 2001. Morphological and molecular characterization of Pratylenchus arlingtoni n. sp., P. convallariae and P. fallax (Nemata: Pratylenchidae). Nematology. V. 3 p. 607-618.

Viruses

Viruses of citrus, vegetables, ornamentals and stone fruits

Accomplishment:  A novel tobamovirus was isolated from symptomatic hibiscus in Florida nursery stock and landscape plantings, characterized and subsequently recognized as a new species, Hibiscus Latent Fort Pierce Virus (HLFPV). Detection strategies have been developed, tested and transferred.

Impact:  HLFPV and a recently discovered tobamovirus from Singapore are the first well-characterized tobamoviruses known to infect malvaceous hosts, including cotton, okra and kenaf. This discovery has extended virological knowledge and necessitates a revision of the tobamovirus genus to accommodate the addition of a new subgroup.

Additional Information:  Major collaborations have included University of Florida-Citrus Research and Education Center, Florida Department of Agriculture and Consumer Services Division of Plant Industry and APHIS-PPQ.

Documentation:

Kamenova, I., Adkins, S. 2004. Comparison of detection methods for a novel tobamovirus isolated from Florida hibiscus. Plant Disease. V. 88 p. 34-40.

Adkins, S., Rosskopf, E.N. 2002. Key West nightshade, a new experimental host for plant viruses. Plant Disease. V. 86 p. 1310-1314.

R.H. Brlansky, M.E. Hilf, P.J. Sieburth, W.O. Dawson, P.D. Roberts and L.W. Timmer. 2005. 2005 Florida Citrus Pest Management Guide: Tristeza. PP-181, Plant Pathology Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida.

Hughes, H., Gottwald T. R., and Yamamura, K. 2001. Survey methods for assessment of citrus tristeza virus incidence in urban citrus populations. Plant Disease 86:367-372.

Viruses of floral and woody ornamental plants

Accomplishment:  Using serological and molecular technologies, ARS scientists determined the identity of and performed the initial characterization of several of new and emerging viruses of floral and woody ornamental plants, and produced reagents and tools for their detection and diagnosis. 1. A Pea mosaic strain of Bean yellow mosaic potyvirus was identified for the first time by serology, cloning and sequencing; this virus was found in a mixed infection with broad bean wilt fabavirus causing a mosaic disease in Verbena. 2. Two new potyviruses infecting the orchid Spiranthes were discovered and portions of their genomes were sequenced. 3. Two additional potyviruses, one associated with flower break symptoms in New Guinea Impatiens and the other associated with leaf mosaic in Omphalodes, were discovered and partially characterized, and their 3’ terminal genomes were sequenced. 4. The complete nucleotide sequence of a Gladiolus isolate of Bean yellow mosaic virus (BYMV) was determined. 5. Strains of Alternanthera mosaic potexvirus (AltMV) infecting creeping phlox and trailing Portulaca were also identified, with one isolate from phlox completely cloned and sequenced and the 3’ regions of two additional phlox and one Portulaca isolate sequenced. 6. A previously uncharacterized carlavirus from creeping phlox has also been completely cloned and sequenced, and characterization continues. 7. A tobamovirus isolate infecting Petunia has been partially cloned and sequenced, and shown to be distinct from other characterized isolates. 8. Polymerase chain reaction tests were developed for detection of AltMV, a newly identified phlox carlavirus, a presumptive carmovirus from Angelonia, and the Petunia tobamovirus. 9. Calla lily chlorotic spot virus and Calla lily latent virus were identified in diseased calla lily plants and were shown to be new, distinct tospovirus and potyvirus members, respectively; carnation mottle virus was identified for the first time in diseased calla lilies showing yellow mottling, light yellow spots and mosaic symptoms. 10. Amaryllis mosaic, Lycoris mild mosaic viruses and two yet to be named Lycoris potyvirus and Bacopa ilarvirus are being investigated 11. Other unknown or previously undescribed emerging viruses causing diseases in Amorphophallus, Angelonia, Bacopa, Cyrtanthus, Lachenalia, Nandina, Ornithogalum, Phlox, Scaveola, Tricyrtis and Viola are currently under investigation.

Impact:  Other than natural or engineered resistance (which is rare), the prevention of disease through selection of pathogen-free or pathogen-indexed plants is the best method of controlling viral diseases. Identification of these new and emerging viruses of ornamentals and the subsequent development of diagnostic reagents for virus detection protocols will allow U.S. floriculture companies and other growers to test propagation stock in order to select healthy plants, resulting in increased productivity, quality, and customer satisfaction. Improved diagnostic reagents will also help protect domestic crops from existing and foreign pathogens. Coat protein and antisense RNA constructs were used to transform Gladiolus to investigate virus resistance.

Additional Information:  Three Specific Cooperative Agreements funded through the Floral and Nursery Research Initiative support research on other viruses of interest to the floral industry. In addition, under an SCA with the University of California-Riverside, a tymovirus affecting Diascia, Nemesia, and Verbena has been partially characterized, an antiserum was produced for detection, and experiments were conducted to identify the optimum tissues to sample for reliable detection. The host range of the virus was examined, and the double-stranded RNA profile of the virus determined, allowing specific detection of the tymovirus in mixed infections. A second virus is also being characterized. Another SCA, with Ohio State University, has examined the distribution of a tobamovirus in Petunia, and the environmental conditions that lead to greatest reliability of detection by ELISA. The third SCA, with Oregon State University, has focused on the detection of a potyvirus in Verbena, and the ability to detect the virus during tissue culture propagation. The virus was identified as a pea mosaic virus isolate of Bean yellow mosaic virus, and it was found to be reliably detected by an ELISA using a cross-reactive monoclonal antibody previously developed by ARS. Work on characterization of a carmovirus in Angelonia is being carried out in collaboration with ARO, Bet Dagan, Israel.

Documentation:

Hammond, J. and Jordan, R.L. 2001. Potyviruses. in: Encyclopedia of Plant Pathology. John Wiley & Sons, New York. pp. 792-800.

Hammond, J., Hammond, R. 2003. The complete nucleotide sequence of Bean yellow mosaic virus isolate BYMV-GDD and comparison to other potyviruses. Archives of Virology. v.148 p. 2461-2470.

Nemchinov, L.G., Hammond, J., Jordan, R.L., Hammond, R. 2004. The complete nucleotide sequence, genome organization, and specific detection of Beet mosaic virus. Archives of Virology. v.149 p. 1201-1204.

Kamo, K.K., Gera, A., Cohen, J. Hammond, J., Blowers, A., Smith, F., van Eck, J. 2005. Transgenic Gladiolus plants transformed with either the bean yellow mosaic virus coat protein or antisense RNA. Plant Cell Reports v.23:654-663.

Hsu, H. T., Ueng, P. P., Chu, F. H., Ye, Z., and Yeh, S. D., 2000. Serological and molecular characterization of a high temperature-recovered virus belonging to tospovirus serogroup IV. J. Gen. Plant Pathol. 66:167-175.

Chen, C.C., Chen, Y.K., and Hsu, H.T., 2000. Characterization of a virus infecting lisianthus. Plant Disease 84:506-509.

Hsu, H. T., Barzuna, L., Hsu, Y. H., Bliss, W., and Perry, K. L., 2000. Identification and subgrouping of Cucumber mosaic virus with mouse monoclonal antibodies. Phytopathology 90:615-620.

Gera, A., Hsu, H.T., Cohen, J., Watad, A., Beckelman, and Hsu, Y.H.,  2000. Effect of cucumber mosaic virus monoclonal antibodies on virus infectivity and transmission by Myzus persicae   J. Plant Pathol. 82:119-124

Aebig, J.A., Kamo, K., and Hsu, H.T.,  2005. Biolistic inoculation of gladiolus with cucumber mosaic cucumovirus. Journal of Virological Methods. J. Virol. Methods. 123:89-94.

Exotic viruses of sweet potatoes, small fruits (gooseberries and currants) and stone fruits

Accomplishment:  ARS scientists demonstrated that Sweet potato virus disease (SPVD) could be caused by a synergistic interaction of Sweet potato chlorotic stunt virus (SPCSV) with any one of several different potyviruses that infect sweet potatoes, not just sweet potato feathery mottle virus (SPFMV). Furthermore, the research showed that the severity of the disease symptoms varied with the individual potyvirus component of the mixed infection. This was the first report of potyviruses, other than strains of SPFMV, interacting with SPCSV to cause SPVD.

Impact:  PCSV is not known to occur in commercial productions of sweet potato in the United States. However, three potyviruses are now known to be present. All three of the viruses were shown to interact with SPCSV to produce SPVD. One of these viruses, Sweet potato vein mosaic virus, was shown to cause an even more severe form of SPVD than that produced by the classical SPFMV/SPCSV mixed infection. Therefore, if SPCSV were introduced into the United States in infected germplasm, the economic impact to sweet potato production could be serious. This new knowledge will be valuable to breeders who are developing new varieties with resistance to SPVD.

Documentation:

Lotrakul, P., Valverde, R., Clark, C., Hurtt, S., Hoy, M. 2002. Sweet potato leaf curl virus and related geminiviruses in sweetpotato. Acta Horticulturae 583: 135-141.

Grapevine rootstock stem lesion complex and young vine decline of grape

Accomplishment:  Graft-inoculations onto test plants of Cabernet Sauvignon scions on hybrid grape rootstocks, those resistant to Phylloxera strain B and used commercially in California vineyards, served as a differential host range to identify a lethal virus strain and several lethal GTAs as part of the Grapevine rootstock stem lesion complex.  The lethal virus strain in Redglobe (RG) table grape was cloned and sequenced and identified as a variant of Grapevine leaf roll associated virus 2 (GLRaV-2); it was designated as GLRaV-2RG and killed graft-inoculated test plants on rootstocks 3309C, 5BB, 5C, 1103P and 1616C. An RT-PCR assay was developed for GLRaV-2RG and was tested on diseased wine grape samples; ca. 20% of them tested positive.  Results of three test trials identified six additional grape sources with lethal GTAs. Their molecular characterizations are in progress.

Impact:  A rapid assay for only GLRaV-2RG has been developed and three new biological indicators have been identified to detect all lethal GTAs discovered to date. They are 3309C, 5BB and 5C. These additions will be proposed for inclusion in the California grapevine clean stock program.

Additional Information:  The discovery of new GTAs in grape was achieved through collaboration with Dr. Adib Rowhani (UC Davis) and various postdoctoral associates funded through the Improvement Advisory Board, the California Competitive Grant Program for Research in Viticulture and Enology, and a CPGR Specific Cooperative Agreement (SCA) with PI Adib Rowhani. Rootstock trials are conducted at the Armstrong Tract in collaboration with the Department of Plant Pathology, UC Davis.

Documentation:

Rowhani, A., Zhang, Y.-P., Golino, D.A., and Uyemoto, J.K. 2000. Isolation and partial characterization of two new viruses from grapevine. Proceedings 13th Meeting International Council for Virus and Virus-like Diseases of Grapevines. Adelaide, Australia. pp. 83-84.

Uyemoto, J. K., Rowhani, A., Luvisi, D., and Krag, C.R. 2001. Discovery of a new   

closterovirus in Redglobe grape causing decline of grafted plants. California 

Agriculture 55(4): 28-31.

Uyemoto, J.K. and Rowhani, A. 2003. Discovery of different grapevine sources with graft- transmissible agents causing union-incompatibility on sensitive rootstocks.

Proceedings 14th International Council for Virus and Virus-like Diseases of

Grapevines. Locorotondo, Italy. pp. 139-140.

Rowhani, A., Uyemoto, J.K., Golino, D.A., and Martelli, G.P. 2005. Pathogen testing and certification of Vitis and Prunus species. Annual Review of Phytopathology 43:  (accepted).

Westphal, A., Browne, G.T., and Schneider, S. 2002. Evidence for biological nature of the grape replant problem in California. Plant and Soil 242:197-203.

Discovery Area 2:  Identification or Genetic Characterization of Known Pathogens or New Strains of Known Pathogens

Prokaryotic Plant Pathogens

Floral and woody ornamental plant prokaryotes

Accomplishment:  ARS scientists discovered the association of Xylella fastidiosa with disorders of three hosts and produced the first report of bacterial leaf scorch in a bonsai tree. ARS scientists also demonstrated the association of X. fastidiosa with a leaf-scorch disorder in Japanese beech bonsai and in black oak in the United States, as well as the causal role of X. fastidiosa in oleander leaf scorch and the presence of the disease in various locations in Texas. In order to determine how X. fastidiosa strains from alternative hosts are related genetically to other hosts, ARS scientists first isolated X. fastidiosa from its alternative hosts (porcelain berry and wild grape) and then determined their genetic relationships with each other and with strains from grape, peach, plum, oak, mulberry, maple and oleander. These alternative host strains of X. fastidiosa were discovered to be more closely related to the oak strain than the grape strain.

ARS scientists also determined that symptomless geranium plants infected with Ralstonia solanacearum race 3 biovar 2 (R3bv2) release easily detectable numbers of bacteria from their roots; this discovery is being developed into a rapid field screening method.

Impact:  The finding that X. fastidiosa is associated with high-valued bonsai is significant because it is an important step leading to the control of the disorder and preservation of the horticultural masterpiece. The discovery is also of great value for regulatory officials when bonsai plants are moved between countries, particularly from the United States to Asian countries where X. fastidiosa has not been found. The findings that X. fastidiosa is associated with black oak and causes oleander leaf scorch in Texas expand the host range of the bacterium in economically important landscape tree species, and extend the geographic range of this important bacterial disease, respectively. The phylogenetic study suggests that alternative hosts may play an important role in the spread of X. fastidiosa to economically important hosts such as oak by serving as a reservoir of inoculum. Removal of these alternative host plants may be important for disease control.

The examination of bacterial wilt-host relationships with Ralstonia solanacearum Race 1 as a model system has led to the screening of bactericides for their ability to protect geranium plants from infection. The beneficiaries of the Ralstonia research are the geranium production industry, the hundreds of U.S. greenhouse ornamental growers that grow and sell geraniums, and APHIS-PPQ, the regulatory agency responsible for developing policies that exclude and eradicate this Select Agent. Even though strict sanitation procedures have been implemented in geranium production facilities, a low cost effective protectant would greatly benefit the $300 million/year geranium industry and will help safeguard the $1.2 billion/year U.S. potato industry.

Additional Information:  Two Specific Cooperative Agreements (SCA) funded through the Floral and Nursery Research Initiative support research on Ralstonia solanacearum. One SCA with the University of Wisconsin-Madison and addresses research on the release of bacteria from symptomless plants; the other is with the University of Florida and examines bacterial wilt-host relationships.

Documentation:

Huang, Q., Li, W. and Hartung, J. S. 2003. Association of Xylella fastidiosa with leaf scorch in Japanese beech bonsai. Canadian Journal of Plant Pathology 25: 401-405.

Huang, Q. and Sherald, J. L. 2004. Isolation and phylogenetic analysis of Xylella fastidiosa from its invasive alternative host, porcelain berry. Current Microbiology 48: 73-76.

Huang, Q. 2004. First report of Xylella fastidiosa associated with leaf scorch in black oak in Washington, D. C. Plant Disease 88: 224.

Huang, Q., Brlansky, R. H., Barnes, L., Li, W. and Hartung, J. S. 2004. First report of oleander leaf scorch caused by Xylella fastidiosa in Texas. Plant Disease 88: 1049.

Fungi

Phytophthora spp. of strawberry, nut trees (walnuts/almonds), stone fruit and grape

Accomplishment: ARS scientists have characterized genetic (AFLP) and pathogenic diversity of P. cactorum and P. citricola, two pathogens that cause widespread damage on almond, strawberry, and many other California crops.

Impact: The pathogenic and genetic characterizations of populations of Phytophthora cactorum and P. citricola from diverse hosts will provide a clear picture of the population structure of these important pathogens. This is an essential component in an effective cultivar and rootstock breeding programs for Phytophthora resistance.

Documentation:

Browne, G.T., Viveros, M.A. 2005. Effects of phosphonate and mefenoxam treatments on development of perennial cankers caused by two Phytophthora spp. on almond. Plant Disease 89:241-249.

Browne, G.T., Wilcox, W.F., Latore. 2005. Phytophthora crown and root rot. In: Compendium of Grape Diseases. The American Phytopathological Society, St. Paul.

Nematodes

Soybean cyst nematode

Accomplishment:  Soybean cyst nematode is able to overcome all known sources of resistance. A genetic linkage map was developed for soybean cyst nematode and markers for virulence to some sources of resistance were located on the map.

Impact:  The genetic linkage map will provide for map-based cloning of virulence genes of soybean cyst nematode and will provide the framework for whole genome sequencing. This genetic linkage map is the second one developed for a plant-parasitic nematode and the first developed for SCN. This groundbreaking work is the most important research done thus far to understand the genetics of SCN, especially as it relates to parasitism and ability to defeat plant resistance.

Additional Information: Collaborative efforts with colleagues from the University of Illinois and outside funding provided personnel and resources to produce the first genetic linkage map of soybean cyst nematode and to place markers for virulence on the linkage map.

Documentation:

Atibalentja, N., Bekal, S., Domier, L. L., Niblack, T. L., Noel, and Lambert, K. N. A genetic linkage map of the soybean cyst nematode, Heterodera glycines. Molecular Genetics and Genomics. (Accepted 12/29/2004).

Viruses

Rhizomania of sugarbeet and other viruses of sugarbeet, lettuce, tomato, cucurbits, and strawberry

Accomplishment:  Beet necrotic yellow vein virus (BNYVV) is the causal agent of rhizomania disease of sugar beet. In 2002-2004 in the Imperial Valley of California, resistant sugar beet cultivars containing the Rz1 allele were severely affected by rhizomania and contained elevated virus levels. Distinct BNYVV isolates from Imperial Valley (IV-BNYVV) were identified from infected sugar beet roots by single local lesion isolation. Because these isolates do not contain RNA-5 as determined by RT-PCR and the banding patterns of single-strand conformation polymorphism analyses, we concluded that the resistance-breaking BNYVV isolates had likely evolved from the original existing A-type. The pathogenicity of IV-BNYVV isolates was studied, and PCR products from coat protein (RNA-2) and P-25 protein (encoded by BNYVV-RNA-3, involved in symptom expression) of IV-BNYVV isolates were sequenced. Sequence alignments from both coat protein and P-25 protein revealed only minor amino acid changes compared to the existing A-type of California BNYVV isolates.

Impact: This research demonstrated that minor genetic changes may have allowed the A-pathotype to overcome Rz1-mediated resistance. This knowledge is critical to development of new strategies for control of rhizomania throughout the world, through both traditional breeding and biotechnology. Furthermore, this research has provided important knowledge that has implications for prolonging the useful life of Rz- resistant germplasm in areas where resistance has not yet been overcome by virus evolution. Such rapid (only a few sugarbeet crops after beginning use of resistant germplasm) evolution of resistance-breaking pathotypes indicates it may be necessary to moderate the use of resistant germplasm in order to reduce the rate of emergence of resistance breaking pathotypes in other areas.

Documentation:

Wisler, G.C, Lewellen, R.T., Sears, J.L., Wasson, J., Liu, H.-Y. and Wintermantel, W.M. 2003. Interactions between Beet necrotic yellow vein virus and Beet soilborne mosaic virus in sugar beet. Plant Disease 87: 1170-1175.

Liu, H. Y., Sears, J. L., and Morrison, R. H. 2003. Isolation and characterization of a carom-like virus from Calibrachoa plant. Plant Dis. 87:167-171.

Wintermantel, W.M. 2005. Co-infection of Beet mosaic virus with beet yellowing viruses leads to increased symptom expression on sugarbeet. Plant Disease 89: 325-331.

Liu, H. Y., Sears, J. L., and Lewellen, R. T. 2005. Occurrence of resistance-breaking Beet necrotic yellow vein virus of sugar beet. Plant Disease. (in press/accepted for publication December, 2004).

Viruses of citrus, vegetables, ornamentals and stone fruits

Accomplishment: Considerable progress was made on two important insect-transmitted pathogens of citrus. The brown citrus aphid (BCA) is the most efficient vector of severe stem pitting isolates of citrus tristeza virus (CTV). ARS has separated several mild and severe CTV sub-isolates from mild field isolates of CTV. When sweet orange and Mexican limes were re-inoculated with known isolates, new and apparently recombinant forms were subsequently re-isolated. There are indications that the BCA may be selectively transmitting one strain versus others and/or the aphid serves as a bottleneck in separating virus population components. In addition, new genotypes of CTV were discovered.

Impact: This knowledge increases our understanding of the genetic diversity of CTV, provides data for improved control of CTV-caused diseases, will lead to better detection technologies, and will result in better screening of citrus germplasm.

Documentation:

Brlansky, R.H., Damsteegt, V.D., Howd, D.S., and Roy, A. 2003. Molecular analyses of Citrus tristeza virus subisolates separated by aphid transmission. Plant Disease 87:397-401.

Discovery Area 3:  Development of New Methods for Identifying or Classifying Pathogens

Prokaryotic Plant Pathogens

Structural and functional genomics of potato, strawberry, grapevine, jujube, walnut, bean, carrot, peach, cherry, pear, and almond pathogens: Ultrasensitive PCR assays

Accomplishment:   Highly sensitive means for detection of phytoplasmas and better understanding of phytoplasma ecology were needed for disease management, quarantine, and curbing disease spread. ARS scientists developed novel ultrasensitive nested-PCR assays for universal (all phytoplasmas), broad (specific group) and specific (specific subgroup) detection of phytoplasmas. New insights of phytoplasma ecology and biodiversity were achieved through use of these new molecular tools. Ultrasensitive PCR using universal primers permitted for the first time the detection of a broad array of phytoplasmas associated with plants and insect vectors. In mixed infections, specific nested-PCR allowed the detection of secondary phytoplasmas (often present in unusually low concentrations) associated with many economically important woody plant species and some herbaceous plants; they were essentially undetectable by PCR assays with universal primers. This ultrasensitive method allowed for the first time the detection of multiple phytoplasmas in a single plant, thereby unraveling the complexity of phytoplasma diversity and ecology. Thus far, these molecular tools have been used to identify more than 20 new diseases and several new phytoplasma strains.

Impact: The development of “universal” primer pairs and nested-PCR assays permitted the broadest and most sensitive detection of phytoplasmas and made it possible to develop the first comprehensive classification of phytoplasmas based on 16S rDNA sequence analysis. These tools enabled researchers to detect and identify phytoplasmas that previously could not be detected. The nested PCR technologies for phytoplasma detection have been adopted by a diagnostic company (Agdia, Inc.) and by APHIS, as well as by research groups and diagnostic laboratories worldwide. This accomplishment has expanded the current knowledge of and changed the concepts of phytoplasma diversity and ecology.

Additional Information:  A Trust Fund granted by AGDIA Inc. facilitated the design of PCR primers for the detection of phytoplasmas.

Documentation:

Abou-Jawdah, Y., Karakashian, A., Sobh, H., Martini, M., and Lee, I.-M. 2002. An epidemic of almond witches’-broom in Lebanon:  Classification and phylogenetic relationships of the associated phytoplasma. Plant Dis. 86:477-487.

Lee, I.-M., Martini, M., Bottner, K. D., Dane, R. A., Black, M., and Troxclair, N. 2003. Ecological implications from a molecular analysis of phytoplasmas involved in an aster yellows epidemic in various crops in Texas. Phytopathology 93:1368-1377.

Lee, I.-M., Bottner, K. D., Munyaneza, J. E., Secor, G.A., and Gudmestad, N. C. 2004. Clover proliferation group (16SrVI), subgroup A (16SrVI-A) phytoplasma is probable causal agent of potato purple top disease in Washington and Oregon. Plant Dis. 88:429.

Liu, Q., Wu, T., Davis, R.E., and Zhao, Y. 2004 First report of witches’-broom disease of Broussonetia papyrifera and its association with a phytoplasma of aster yellows group (16SrI). Plant Dis. 88:770.

Montano, H.G., Davis, R.E., Dally, E.L., Pimentel, J.P., and Brioso, P.S.T. 2000. Identification and phylogenetic analysis of new phytoplasma from diseased chayote in Brazil. Plant Dis. 84:429-436.

Structural and functional genomics of potato, strawberry, grapevine, jujube, walnut, bean, carrot, peach, cherry, pear, and almond pathogens: Molecular systematics of phytoplasmas

Accomplishment:  To date, phytoplasmas cannot be cultured in cell-free media. The traditional classification and taxonomy for walled bacteria based on biochemical properties and DNA-DNA homology is not applicable for phytoplasmas. Lack of rapid means for identification and classification of a broad array of phytoplasmas has hindered studies on the ecology of phytoplasmas and on the epidemiology of phytoplasmal diseases. ARS has evaluated the usefulness of conserved genes including 16S rRNA, ribosomal protein (rp), and secY, for classification of phytoplasmas. Broad-spectrum primers were designed for amplification of these genes from phytoplasmas. We devised and constructed a comprehensive classification scheme for a broad array of phytoplasmas (consisting of 15 phytoplasma groups and more than 50 subgroups) based on RFLP analysis of PCR-amplified 16S rDNA, rp, and secY gene sequences. This new scheme represents the most comprehensive classification system available that allows for the identification of numerous phytoplasmas worldwide. Three ‘Candidatus Phytoplasma species’ have been proposed.

Impact: This accomplishment provided a rapid approach for identification of phytoplasmas that was adopted by scientists worldwide and a system recognized internationally peers as a major breakthrough for classification of phytoplasmas. As a result, the phytoplasma research field has dramatically expanded. The system is currently used by the National Center for Biotechnology Information (NBCI)/GeneBank for classification of phytoplasmas. This accomplishment has generated many collaborative research projects both nationally and internationally. The taxonomic proposal was a key breakthrough that launched phytoplasma taxonomic speciation and was adopted by the International Committee of Systematic Bacteriology Subcommittee on the Taxonomy of Mollicutes. Thus far, genus Phytoplasma has been officially proposed, and more than 20 Candidatus phytoplasma species have been published. The much-needed genomics information is also being used by California Department of Agriculture, in collaboration with ARS, to develop real-time PCR protocols for early detection of corn stunt epidemics; in California; an outbreak of the disease in 2002 caused damage exceeding $5 million in Kings County alone. On the practical aspect, these new discoveries have improved strain and species differentiation and classification, and will lead to development of new tools for pathogen detection and identification.

Documentation:

Montano, H. G., Davis, R.E., Dally, E.L., Hogenhout, S., Pimentel, J.P., and Brioso, P.S.T. 2001. ‘Candidatus Phytoplasma brasiliense’, a new phytoplasma taxon associated with hibiscus witches’-broom disease. Int. J. Syst. Evol. Microbiol. 51:1109-1118.

Lee, I.-M., Gundersen-Rindal, D. E., Davis, R. E., Bottner, K. D., Marcone, C., and Seemüller, E. 2004. ‘Candidatus Phytoplasma asteris’, a new phytoplasma taxon associated with aster yellows and related diseases. Int. J. Syst. Evol. Microbiol. 54:1037-1048.

Lee, I.-M., Martini, M., Marcone, C., and Zhu, S. F. 2004. Classification of phytoplasma strains in the elm yellows group (16SrV) and proposition of ‘Candidatus Phytoplasma ulmi’ for the phytoplasma associated with elm yellows. Int. J. Syst. Evol. Microbiol. 54:337-347.

Jomantiene, R., Davis, R.E., Valiunas, D., Alminaite, A., and Staniulis, J. 2002. New group 16SrIII phytoplasma lineages in Lithuania exhibit rRNA interoperon sequence heterogeneity. Europ. J. Plant Pathol. 108:507-517.

Lee, I.-M., Davis, R.E., and Gundersen-Rindal, D.E. 2000. Phytoplasma: Phytopathogenic Mollicutes. Annu. Rev. Microbiol. 54:221-255.

Exotic prokaryotic pathogens of citrus, cereals, watermelon, grape, shade trees, potato, tomato, grass seed, and rice

Accomplishment:  Real-time PCR assays for rapid, routine detection of several high-risk bacterial pathogens were developed. A real-time BIO-PCR protocol was developed for detection of the APHIS Select Agent Ralstonia solanacearum race 3, biovar 2 in asymptomatic potato tubers within 48 hours. A direct (i.e., without DNA extraction) real-time PCR assay was developed for rapid, on-site diagnosis of Pierce’s disease in grape, leaf scorch in shade trees, and citrus variegated chlorosis (CVC) in citrus. The method employs dry beads containing all PCR ingredients, including Taq polymerase, primers and probe for use with a portable PCR unit. The citrus canker bacterium Xanthomonas campestris pv. citri was detected in confiscated leaf and fruit samples on-site at San Francisco (SFO) and Los Angeles International (LAX) airports, by use of a portable PCR unit and newly developed real-time PCR primers and probe. A real-time BIO-PCR assay was developed for detecting Acidovorax avenae subsp. avenae in rice seeds. Over 50 cultures of Russian bacteria were received, including Pseudomonas atrofaciens, X. oryzae pv. oryzae and R. solanacearum race 3, biovar 2 from present and past projects.

Impact: The real-time BIO-PCR assay for R. solanacearum bv2 is being utilized in Russia, Turkey, and parts of Europe for routine assays of potato tubers. Our real-time PCR assay containing dry beads with all PCR ingredients is available commercially through Cepheid, Sunnyvale, CA for rapid, 1-2 hour on-site detection of the devastating exotic CVC bacterium in citrus, Pierce’s disease in grape, and leaf scorch disease in many shade trees. When these diseases are diagnosed before they become established, control is achievable. Use of the real-time PCR assay for X. citri has demonstrated that real-time PCR can be used on-site at U.S. Ports in a routine manner and has established that citrus canker is entering California daily through SFO and LAX airports. This shows that molecular-based detection technologies can be used by port inspectors for rapid, on-site identification of diseased plants brought in by airplane passengers and in commercial shipping containers. By obtaining several cultures from past and present programs in Russia we have made available cultures to be fingerprinted and added to a database of high-risk plant pathogens. Making these rapid, specific PCR assays for high-risk pathogens available has significantly improved the security of U.S. agriculture. Moreover, the BIO-PCR method was patented (U.S. patent no. 6,410,223 issued on 25 June 2002) and is being used in many routine assays in several diagnostic and research fields, including food safety and environmental studies.

Additional Information:  Collaborative research with CDFA and APHIS, SFO facilitated the ability to obtain current and archived citrus canker leaf and fruit samples at LAX and SFO.   A scientist from Turkey with support from NATO spent a one-year sabbatical with ARS to assist in developing the real-time PCR assay for detecting R. solanacearum biovar 2 in potato. Cepheid, Sunnyvale, California collaborated in developing the rapid, on-site PCR assay for Pierce’s disease and CVC by providing dry beads for real-time PCR free of charge. A Russian Scientist provided the cultures from Russia; this work in Russia would not have been possible without support from an ISTC grant; ISTC also supported a Russian scientist to spend six months at Ft. Detrick characterizing bacteria.

Documentation:

Schaad, N.W., Frederick, R.D., Shaw, J., Schneider, W.L., Hickson, R., Petrillo, M.D., and Luster, D.G. 2003. Advances in Molecular-Based Diagnostics in Meeting Crop Biosecurity and Phytosanitary Issues. Ann. Rev. Phytopathol. 41:305-324.

Ozakman, M. and Schaad, N.W. 2003. A real-time BIO-PCR assay for detection of Ralstonia solanacearum race 3, biovar 2, in asymptomatic potato tubers. Can. J. Plant Pathol. 25:232-239.

Schaad, N. W., Postnikova, E., Lacy, G., Fatmi, M., and Chung-Jan, C. 2004. Xylella fastidiosa subspecies: X. fastidiosa subsp. piercei, subsp. nov., X. fastidiosa subsp. multiplex subsp. nov., and X. fastidiosa subsp. pauca subsp. nov. Syst. Appl. Micro. 27:290-300.

Song, W. Y., Kim, H. M., Hwang, C. Y., and Schaad, N. W. 2004. Detection of Acidovorax avenae ssp. avenae in rice seed using BIO-PCR. J. Phytopathol. 152: 667-676.

Mateeva, I.E.V., Pekhtereva, E. SH., Polityko, V.A., Ignatov, A.N., Nikolaeva, E.V., and Schaad, N.W. 2003. Distribution and virulence of Pseudomonas syringae pv. atrofaciens, causal agent of basal glume rot, in Russia. pp. 97-105. In: Pseudomonas syringae and related pathogens, N.S. Lacobellis, et al., (eds). Kluwer Academic Publishers, Dordrecht, The Netherlands (Book).

Crown gall and deep bark canker (DBC) of nut trees (walnuts/almonds)

Accomplishment:  A real-time PCR detection and quantification system for A. tumefaciens residing in soil has been fully developed. PCR-based detection technologies for the bacterium Brennaria rubrifaciens, the causative agent of deep bark canker of walnut, have also been developed. Impact: Both of these new detection systems will aid in the development of sustainable control strategies for crown gall disease of walnut and almonds and deep bark canker of walnut. In addition, these detection tools are greatly aiding our efforts to examine the ecology of both Agrobacterium and Brennaria under orchard conditions and will aid in walnut breeding programs designed to identify disease resistant selections.

Additional Information: Collaborators in the study of the ecology, biology and control of Agrobacterium tumefaciens include members of the faculty of the University of California-Davis and numerous growers and nursery operators. Research support has been obtained from the Almond Board of California, the Walnut Marketing Board, and the Improvement Advisory Board (IAB) of California.

Documentation:

Robertson, A. E., Wechter, W. P., Denny, T. P., Fortnum, B. A., Kluepfel, D. A. 2004. Relationship between Avirulence Gene (avrA) Diversity in Ralstonia solanacearum and Bacterial Wilt Incidence. Molec. Plant-Microbe Interactions 17: 1376-1384.

Fungi

Molecular and Morphological Systematics of Plant-Pathogenic Fungi

Accomplishment:  Morphological and molecular characters were used to distinguish the ryegrass bunt fungus, Tilletia walkeri, from the Karnal bunt fungus, Tilletia indica. Five key morphological characters were discovered for the identification of Karnal bunt, and a rapid PCR-based test that can be done in any lab with a PCR machine was developed to distinguish the ryegrass bunt from Karnal bunt. Online morphological identification resources have also been made available for species of Tilletia in the United States.

Impact:  Methods to accurately identify quarantine significant plant pathogens reduce unnecessary delays and expenses for U.S. grain exporters and allow USDA APHIS to issue accurate phytosanitary certificates. This work has contributed to the accurate identification of species of Tilletia found by APHIS surveys and APHIS PPQ identifiers. Because Karnal bunt can now be accurately identified and its geographic distribution is known, U.S. wheat can be exported worldwide without unnecessary delays or risk of rejection by the importing country. This work also helps protect areas where Karnal bunt is not known to occur by preventing movement of infected grain. Improved methods of identification of both bunt fungi and canker-causing fungi are the long-term objectives for this project.

Additional Information: Collaboration with Washington State University (WSU) contributed to this accomplishment. WSU provided specimens and expertise in the systematics of wheat bunt fungi. APHIS scientists also facilitated this work by providing specimens and information. Karnal bunt data has been incorporated into the Web-based database of the U.S. National Fungus Collections; the database incorporates data from 700,000 specimens, accurate scientific names and plant hosts and distribution for fungi in the United States, and over 500,000 reports of plant-associated fungi throughout the world. These data support plant quarantine officials in preventing the entry of invasive fungi estimated at causing $12-15 billion damage annually to crop plants and forests.

Documentation:

Crous, P.W. Van Jaarsveld, A.B., Castlebury, L.A., Carris, L.M., Frederick, R.D., Pretorius, Z.A. 2001. Karnal bunt of wheat newly reported from the African continent. Plant Disease V. 85 p. 561.

Levy, L., Castlebury, L.A., Carris, L.M. Meyer, R. and Pimentel, G. 2001. ITS sequence based phylogeny and PCR-RFLP differentiation of Tilletia walkeri and Tilletia indica. Phytopathology  V. 91 p. 935-940.

Farr, D.F., Castlebury, L.A. and Rossman, A.Y. 2002. Morphological and molecular characterization of Phomopsis vaccinii and additional isolates of Phomopsis from blueberry and cranberry in the eastern United States. Mycologia V. 94 p. 494-504.

Castlebury, L.A., Rossman, A.Y. Jaklitsch, W., Vasilyeva, L.N. 2002. A preliminary overview of the Diaporthales based on large subunit nuclear ribosomal DNA sequences. Mycologia V. 94 p. 1017-1031.

Rossman, A.Y., Aime, M.C. Farr, D.F.. Castlebury, L.A., Peterson, K.R., Leahy, R. 2004. The coelomycetous genera Chaetomella and Pilidium represent a newly discovered lineage of inoperculate discomycetes. Mycological Progress V. 3 p. 275-290.

Soybean rust

Accomplishment:  Research and technology transfer activities enhanced preparedness for the introduction of soybean rust into the United States. The Internal Transcribed Spacer (ITS) region was cloned, sequenced and analyzed from the two Phakopsora species that cause soybean rust. Approximately 80% nucleotide sequence similarity was observed. Utilizing differences within the ITS region, a PCR diagnostic assay was developed that can identify and differentiate between the two Phakopsora species that cause soybean rust. ARS also conducted several training workshops on soybean rust identification for plant disease diagnosticians from the five Regional Plant Disease Diagnostic Centers, as well as for members of the NC-504 committee on soybean rust.

Impact:  Since its initial introduction in November 2004, the ARS PCR assay was used to confirm the presence of the Asian strain of soybean rust in nine continental U.S. states. Previously, the assay had been used to determine the presence of soybean rust in South Africa, Brazil and Paraguay. One of the participants in the ARS soybean rust training workshops was the first person to identify soybean rust in the continental United States. He attributes his ability to identify soybean rust as a direct result of the ARS training. The 800 lines exhibiting some resistance to soybean rust have been released to universities and public breeders to incorporate into their breeding programs. As a result of the success of the molecular diagnostic assay, its developers have been inundated with requests for soybean rust DNA. Several Material Transfer Agreements have been drafted with U. S. and foreign collaborators who wish to conduct PCR assays for soybean rust.  These include Purdue University, Kansas State University, University of Wisconsin, Cornell University, University of Florida, Mississippi State University, Midwest Research Institute, Pioneer Hybrid, Oregon State University, and the Maryland Department of Agriculture.

Additional Information: The United Soybean Board contributed funding in several areas of soybean rust research. The North Central Soybean Research Program (NCSRP) has provided funding for antibody work with soybean rust.

Documentation:

Frederick, R. D., C. L. Snyder, G. L. Peterson, and M. R. Bonde. 2002. Polymerase chain reaction assays for the detection and discrimination of the soybean rust pathogens Phakopsora pachyrhizi and P. meibomiae. Phytopathology 92:217-227.

Pretorious, Z. A., Kloppers, F. J., and Frederick, R. D. 2001. First report of soybean rust in South Africa. Plant Dis. 85:1288.

Sudden oak death

Accomplishment:  ARS scientists developed a PCR-diagnostic assay based on mitochondrial gene sequences for detecting Phytophthora ramorum with a high level of sensitivity and specificity.

Impact:  Under a Material Transfer Agreement, PCR primers based on the coxII gene were transferred to the California Dept. of Food and Agriculture for detection of P. ramorum and the detection technology is now available to end users such as APHIS.

Documentation:

Martin, F. N., Tooley, P. W., and Blomquist, C. 2004. Molecular detection of Phytophthora ramorum, the causal agent of sudden oak death in California, and two

additional species commonly recovered from diseased plant material. Phytopathology

94:621-631.

Tooley, P. W., Kyde, K. L., and Englander, L. 2004. Susceptibility of selected ericaceous ornamental host species to Phytophthora ramorum. Plant Disease 88:993-999.

Soilborne fungal pathogens of alfalfa and dry beans

Accomplishment:  A sequence characterized amplified region (SCAR) DNA marker, OPC7₁₃₃₂, was developed. The marker can detect the soilborne root rot pathogen Aphanomyces euteiches in infected plants and infested field soil. Another SCAR marker (PSC12₄₉₉) was developed; it can detect the soilborne fungus Phoma sclerotioides, the causal agent of brown root rot of alfalfa, in infected plants and infested field soil samples.  Real-time quantitative PCR assays have been developed that can quantify A. euteiches and Phytophthora medicaginis in infected alfalfa plants. Tests have been conducted using several different alfalfa populations to determine the correlation between integer disease severity ratings and the amount of pathogen DNA detected in infected plants. The two real-time PCR assays have been used in a multiplex reaction to examine the population dynamics between A. euteiches and P. medicaginis in alfalfa plants co-infected with both pathogens.

Impact: The ability to detect the presence of pathogens in fields prior to planting provides growers with decision tools for selecting cultivars that have appropriate disease resistance profiles. At least three weeks are required for detecting A. euteiches in soil with conventional microbiological approaches; in the case of P. sclerotioides, 60 days are required to unambiguously detect the pathogen in soil samples. The SCAR markers OPC7₁₃₃₂ and PSC12₄₉₉ can detect the presence of these pathogens in soil in a single day. The rapid response time for results based on these PCR assays allows for timelier and accurate decision making on the part of growers with respect to choosing cultivars for production in infested fields. The real-time quantitative PCR assay provided a reliable method for distinguishing among commercial alfalfa varieties for resistance to A. euteiches based on the analysis of bulked plant samples. Previously, standard tests for evaluating resistance to A. euteiches and P. medicaginis required the evaluation of many individual plants (³ 200). The real-time PCR assays will accelerate the process of screening populations by providing a means for determining resistance levels based on an analysis of bulked plant samples. Plants that appear phenotypically similar can be discriminated based on the results of these real-time PCR assays. Commercial seed companies have adopted these technologies to provide additional, more quantitative data to demonstrate the performance of plant populations in support of applications for the review of new varieties. The two real-time PCR assays can be used to simultaneously select plants for resistance to A. euteiches and P. medicagnis. These assays can also be used to study microbial population dynamics in mixed infections with much greater accuracy and specificity that can be realized using previously employed techniques such as microscopy and enzyme linked immunosorbent assays (ELISA).

Documentation:

Vandemark, G., Barker, B. 2003. Quantifying Phytophthora medicaginis in susceptible and resistant alfalfa with a real-time fluorescent PCR assay. Journal of Phytopathology. V. 151:577-583.

Larsen, R., Hollingsworth, C., Vandemark, G., Gritsenko, M., Gray, F. 2002. Use of PCR-based markers for the identification of Phoma sclerotiodes causing brown root rot of alfalfa. Plant Disease. V. 86:928-932.

Vandemark, G. J., B. M. Barker and M. A. Gritsenko. 2002. Quantifying Aphanomyces euteiches in alfalfa with a fluorescent polymerase chain reaction assay. Phytopathology. V. 92:265-272.

Nematodes

Molecular and Morphological Systematics of Plant-Pathogenic Nematodes:

Accomplishment:  ARS researchers developed improved nematode identification techniques and organizational frameworks consisting of comprehensive keys and molecular phylogenetic trees. Species identification keys for the economically important cyst nematode avenae group and the complex 111-member stunt nematode group were published. A LSU 28S rDNA phylogenetic tree of lesion nematodes was supplemented with nine more nematode sequences and outgroup taxa to more clearly reflect the expected morphologically based taxonomy. Hsp90, a homologue of the single-copy nematode gene daf-21, was discovered to be a molecular character of potential interest; taxonomically and phylogenetically useful variation was demonstrated in coding and non-coding sequences for several plant-parasitic nematodes. An innovative PCR method led to direct amplification of the Hsp90 gene from single crushed nematodes, and numerous PCR primers and protocols enabled Hsp90 amplification from diverse species of nematodes. A set of protocols for identifying quarantined nematode pests of importance to the North American potato trade was developed. Because accurate identifications can occur only when reliably identified reference specimens are available, the USDA Nematode Collection was maintained, curated, and expanded by adding 5,204 slides and vials from world-wide sources to yield a total collection of 40,079 slides and vials.

Impact:  These most current, accurate and inclusive identification keys for stunt and avenae-cyst nematodes are used by nematode diagnostic and systematics laboratories throughout the world. The new lesion nematode molecular sequences contributed to the alignment needed for the creation of species-specific primers by other researchers for more timely species identification. Hsp90 is gaining in utility as a new phylogenetic marker for different taxa by researchers in Utah and Italy, who are using the Hsp90 primers and methods. Several novel DNA sequences were submitted to GeneBank. Scientists from the United States, Brazil, and Poland have utilized the novel PCR method. Diagnosticians will utilize the potato nematode identification protocols, thereby minimizing trade disputes involving uncertain nematode identifications. Over 1,224 slides from the USDA Nematode Collection were used by other scientists as reference material, and the Web-based Collection database received over 12,000 hits during the five-year period.

Documentation:

Handoo, Z.A., Carta, L.K., Skantar, A.M. 2001. Morphological and molecular characterization of Pratylenchus arlingtoni n. sp., P. convallariae and P. fallax (Nemata: Pratylenchidae). Nematology. V. 3 p. 607-618.

Handoo, Z.A. 2002. A key and compendium to species of the Heterodera avenae group (Nematode: Heteroderidae). Journal of Nematology. V. 34 p. 250-262.

Skantar, A.M., Carta, L.K. 2000. Amplification of HSP90 homologs from plant-parasitic nematodes using degenerate primers and ramped annealing PCR. Biotechniques: V. 29 p. 1182-1185.

Viruses

Exotic viruses of stone fruits and citrus

Accomplishment:  Since the discovery of plum pox(PPV), the most important virus disease of stone fruits, in Pennsylvania in 1999, there has been an effort to develop improved detection capabilities and to understand the pathway of introduction, strain(s) of the virus agent, mechanisms of transmission and the host range. ARS research has established that all isolates found in the United States to date belong to one of two clades of strain D of plum pox potyvirus. A very effective, specific real-time PCR platform was developed for rapid identification of any PPV strain. The potential host range of the virus in wild and ornamental Prunus species has been determined, and the role of endemic orchard aphids in local transmission has been established.

Impact:  Understanding the molecular nature of PPV populations provides guidance on the origin of the viral introductions, pathways of movement, and frequency of mutational change. Determination of fruit as an infectious unit has resulted in new recommendations for disposal of cull fruit. Determination of the potential host range of the Pennsylvania isolates has reduced the quarantine pressure on movement of cherry germplasm and provided insights into what alternative species may be important sources of inoculum influencing the complete eradication of PPV.  The real-time PCR primers and probes developed in our laboratory are being used in the quarantine area in Pennsylvania for rapid, accurate detection of new isolates of PPV.

Documentation:

Schneider, W.L., Sherman, D.J., Stone, A.L., Damsteegt, V.D., and Frederick, R.D. 2004. Specific detection and quantification of Plum pox virus by real-time fluorescent reverse transcription-PCR. Journal of Virological Methods 120:97-105.

Gildow, F., Damsteegt, V., Stone, A., Schneider, W., Luster, D., and Levy, L. 2004. Plum pox in North America: Identification of aphid vectors and a potential role for fruit in virus spread. Phytopathology 94:868-874.

Fatmi, M., Damsteegt, V.D., and Schaad, N.W. 2005. A combined agar-adsorption and BIO-PCR assay for rapid, sensitive detection of Xylella fastidiosa in grape and citrus. Plant Pathology 54:1-7.

Viruses of citrus

Accomplishment:  Research demonstrated the utility of using degenerate primers and PCR to identify genetically distinct components in complex mixtures of Citrus tristeza virus (CTV).

Impact:  These results enhanced regulatory measures concerning identification and management of this pathogen.

Additional information:  Major collaborations during this five year cycle have included University of Florida-Citrus Research and Education Center, Florida Department of Agriculture and Consumer Services-Division of Plant Industry and APHIS-PPQ.

Documentation:

Hilf, M.E., V.A. Mavrodieve and S.M. Garnsey. 2005. Genetic Marker Analysis of a Global Collection of Isolates of Citrus tristeza virus: Characterization and Distribution of CTV Genotypes and Association with Symptoms. Phytopathology, in press.

Viruses of floral and woody ornamental plants

Accomplishment: The complete genomes of four unique viruses infecting geranium were determined: Pelargonium line pattern virus, Pelargonium ringspot virus, Elderberry latent virus, and Pelargonium chlorotic ring pattern virus. Nucleic acid probes were developed for detection of each virus. Virus-specific monoclonal antibodies that detect Carnation necrotic fleck virus and Carnation latent virus were developed and transferred to industry (Agdia, Inc) for use in the detection of these important carnation viruses. Single-chain antibody transgene constructs from broad-spectrum reacting potyvirus monoclonal antibody were created and used to transform model plants to test the expression of antiviral antibodies in transgenic plants as a novel means of virus control. Virus-specific and subgroup-specific mouse monoclonal antibodies for the detection and differentiation of isolates of Cucumber mosaic virus (CMV) were produced and are available for public use through Agdia, Inc. A novel method for inoculation of gladiolus with CMV was developed, and the method enables the efficient evaluation of CMV resistance in gladiolus. Several new and emerging viruses were identified with serological and molecular technologies. During an investigation of the influence of temperature on the ultrastructure of Impatiens necrotic spot virus, a high temperature isolate of a tospovirus, Gloxinia HT-1 tospovirus, was recovered and identified from inoculated plants through a series of propagations at high temperature

Impact: This research has developed the tools, reagents and knowledge that will aid U.S. floriculture companies in establishing effective virus testing protocols that will improve clean stock production for new vegetatively-propagated annuals and perennials. Other than natural or engineered resistance (which is rare), the prevention of disease through the development of more effective means for the detection and identification of plant virus diseases affecting ornamentals, and the utilization of those methods to allow selection of pathogen-free or pathogen-indexed plants, is the best method of controlling viral and bacterial diseases. The identification of new and emerging viruses and the availability of reagents for detection will allow growers to test propagation stock in order to select healthy plants, resulting in increased productivity and quality, and customer satisfaction. Improved diagnostic reagents will also help protect domestic crops from existing and foreign pathogens.

Documentation:

Jordan, R.L. and Guaragna, M.A. 2002. Successful development of monoclonal antibodies to three carnation viruses, using an admixture of only partially purified virus preparations as immunogen, and their use in virus diagnosis. Acta Horticulturae v.568 p.177-184.

Kinard, G.R., Jordan, R.L. 2002. Genome organization of Pelargonium chlorotic ring pattern virus: Further implications for Tombusviridae taxonomy. Acta Horticulturae. v.568 p.17-27.

Zhao, Y., Hammond, J., Tousignant, M.E., Hammond, R.W. 2000. Development and evaluation of a complementation-dependent gene delivery system based on cucumber mosaic virus. Archives of Virology v.145:1-11.

Exotic viruses of sweet potatoes, small fruits (gooseberries and currants) and stone fruits

Accomplishments:  Several molecular protocols were developed and used in combination with biological assays for detection of sweet potato viruses, Gooseberry vein banding virus (GVBV), and two cherry flexiviruses in ARS quarantine indexing programs. Detection of viruses in sweet potato by graft inoculating indicator plants takes up to three months. A PCR assay using degenerate primers has been developed and used for the detection of geminiviruses in in vitro or in vivo plants of Ipomoea spp. A real-time PCR procedure for detection of an exotic virus, sweet potato chlorotic stunt virus, was tested and incorporated into the Ipomoea indexing program.

The previous method for detection of GVBV in Ribes spp. was based on symptom expression in host plants and graft-inoculation to a bio-indicator, neither of which is reliable. A PCR assay has been adapted and used in routine indexing of the virus of gooseberries and currants.

Graft-inoculation of a woody indicator, Kwanzan flowering cherry, was the only method for detection of Cherry green ring mottle virus (CGRMV) in Prunus spp. A one-step RT-PCR assay using consensus primers allows detection of CGRMV and Cherry necrotic rusty mottle virus, a closely related but a previously untargeted flexivirus, in Prunus spp.

Impact: Implementation of these molecular methods will improve the reliability and efficiency of the plant quarantine programs. These newly implemented molecular methods will be used as diagnostic tools to identify the target viruses and their variants. Improved detection/diagnostic methods will help exclude exotic pathogens and therefore provide production security for many important commodities.

Additional Information:  Collaboration with pathologists at Louisiana State University provided the initial methods for extracting nucleic acid from sweet potatoes and amplifying the geminivirus DNA. The ARS scientists improved the tissue extraction methods, applied the PCR assay to in vitro plants, and developed additional primer pairs for detection of sweet potato geminiviruses. Collaboration with LSU was also instrumental in implementing the real-time PCR assay for sweet potato chlorotic stunt virus.

Documentation:

Salih, S., Waterworth, H., Thompson, D. 2001. Role of plant tissue cultures in international exchange and quarantine of germplasm in the United States and Canada. HortScience. 36: 1015-1021.

Kinard, G.R., Waterworth, H.E., and Mock, R.G. 2001. Advances in quarantine testing of temperate fruit tree germplasm at USDA. Acta Horticulturae. 550: 441-445.

Li, R., Salih, S., Hurtt, S. 2004. Detection of geminiviruses in sweetpotato by polymerase chain reaction. Plant Disease 88: 1347-1351.

Viruses of strawberry, blueberry, blackberry and raspberry

Accomplishment:  A new strategy for cloning dsRNA templates was developed and used to efficiently clone viruses from small fruit crops. Cloning was followed by sequencing, developing RT-PCR based laboratory tests for these viruses, and initiating studies on epidemiology. Complete virus sequences were obtained for Strawberry pallidosis, Beet pseudo yellows, Strawberry latent ringspot, Fragaria chiloensis latent virus, Strawberry necrotic shock virus, Blackberry yellow vein, a new closterovirus from raspberry, a new potyvirus from declining blackberry, Black raspberry decline associated virus. Partial sequences were obtained for apple mosaic virus in strawberry, a closterovirus associated with Strawberry chlorotic fleck disease, a new Totivirus from declining blackberry, an insect-like virus from blackberry, a second virus associated with decline in black raspberry, and a virus associated with blueberry fruit drop disease. In addition, five new viruses were identified in mint. Several of these viruses (Strawberry latent ringspot and Tulip virus X have not been reported previously in the United States).

Impact:  The sequence information suggested that whiteflies would be the vector of Strawberry pallidosis. This was confirmed by laboratory experiments and has led to the recommendation that strawberry nurseries control whiteflies to prevent the spread of this virus along with Beet pseudo yellows into plant production nurseries. In areas with high whitefly populations, growers now control whiteflies and the result has been a dramatic decrease in strawberry decline in strawberry production fields in California.  With the RT-PCR tests we showed that strawberry decline in Oregon, Washington and British Columbia is caused primarily by aphid-borne viruses and that Strawberry crinkle virus previously not found in the region previously is now very common. Growers with fields with high aphid populations are now trying to control them, and early results suggest a reduction in virus decline caused by the aphid transmitted strawberry viruses. These tests have been made available to state departments of agriculture and are being used in the certification program for strawberry plant production in California. Raspberry bushy dwarf resistant red raspberry addresses the most serious disease of red raspberries in the Pacific Northwest.

Additional Information:  Funding from commodity groups and cooperation with extension agents has allowed the large scale testing for the strawberry viruses and monitoring the impact of vector control strategies. Development of transgenic raspberries was done in collaboration with Exelixis Plant Sciences Inc. under a CRADA agreement and with funding from the SBIR program.  Fruit quality evaluations of transgenic raspberries were done in collaboration with the Food Sciences Department at Oregon State University and were funded by grants from the Northwest Center for Small Fruit Research.

Documentation:

Hankinson, S.C,  Martin, R.R., Heflebower, R.F. Jr., Rouse, R., Maas, J. 2000. Survey of strawberry viruses occurring in commercial plantings in the state of Maryland, USA. Advances in Strawberry Research 18:25-32.

Martin, R.R., Mathews, H. 2001. Engineering resistance to Raspberry Bushy Dwarf Virus. Acta Hortic. 551:33-37.

Strik, B., Martin, R.R. 2003. Impact of Raspberry bushy dwarf virus on ‘Marion’ blackberry. Plant Dis. 87:294-296.

Tzanetakis, I.E., Martin, R.R. 2004. Complete Nucleotide Sequence of a Strawberry Isolate of Beet pseudo yellows virus. Virus Genes 28:239-246.

Tzanetakis, I.E., Keller, K.E., Martin, R.R. 2005. The use of reverse transcriptase for efficient first- and second-strand cDNA synthesis from single- and double-stranded RNA templates, J. Virol. Methods 124:73-77.

Viruses of lettuce

Accomplishment:  Lettuce dieback disease is caused by a group of tombusviruses known as either Lettuce necrotic stunt virus (LNSV) or Tomato bushy stunt virus (TBSV). The virus causes severe stunting, necrosis and death of lettuce plants, resulting in serious economic losses in romaine and leaf lettuce production in nearly all lettuce production areas of California and Arizona. ARS research led to the identification of a new tombusvirus species responsible for infection of lettuce and tomato and the development of  detection tools for the identification of this virus. In addition, recent studies identified environmental or soil factors that influence disease development; for example, soil salinity has been identified as a factor contributing to elevated incidence of lettuce dieback symptoms when lettuce plants are grown in LNSV-infested soil.

Impact:  The new detection tools were critical to the development and release of resistant germplasm by other ARS collaborators.  The environmental studies are critical to the management of lettuce dieback disease in infested fields, which are common throughout all production areas in California and Arizona.

Additional Information:  This research would not have been possible without the financial support of the California Lettuce Research Board, the California Tomato Commission, and Agdia, Inc. (CRADA). 

Documentation:

Grube, R.C., Wintermantel, W.M., Hand  P., Aburomia, R., Pink, D.A.C., and Ryder, E.J. 2005. Genetic analysis and mapping of resistance to lettuce dieback, a soilborne disease caused by tombusviruses. Theoretical and Applied Genetics 110: 259-268.

Obermeier, C., Sears, J.L., Liu, H.-Y., Schlueter, K.O., Ryder, E.J., Duffus, J.E., Koike, S.T., and Wisler, G.C. 2001. Characterization of distinct tombusviruses that cause diseases of lettuce and tomato in the western United States. Phytopathology 91: 797-806.

Multidisciplinary: The Electron Microscopy Unit

Accomplishment: Through the use of low temperature scanning electron microscopy (LTSEM), pests and pathogens were preserved, thereby allowing researchers to observe pest, pathogens, pests, and host-pathogen relationships without the artifact formation associated with traditional fixation methods. Using these procedures, difficult biological specimens of agricultural importance, including soft bodied forms, were studied in natural behavioral positions with results superior in quality to any fixation technique previously described. For example, one specific accoplishment was the discovery that nematode lips and heads differed greatly in appearance from those observed with chemically fixed specimens this yielded more reliable conclusions about their systematics. Scanning or transmission electron microscopic investigations were involved in the identification or characterization of plant disease vectors, other insects and mites, nematodes, parasites, bacteria, food products and other agriculturally important materials, as well as  the fine structure of numerous disease-causing agents including Onchocerca, Cyclospora, Cryptosporidia, Trichoderma, Monacrosporium, Neospora and Phytophthora.

 Impact: The widespread impact of this project has allowed for increased resolution of structural features used for accurate identification and the study of the association of organisms with the host material. These methodological improvements have impacted the fields of entomology, acarology, nematology and zoology, allowing researchers to rapidly identify and study organisms of major agricultural importance while maintaining biologically significant associations never before seen during the use of conventional preparative methods.

Additional Information:  This multidisciplinary project provided support for research projects within many ARS National Programs (30 collaborators from 16 ARS laboratories) NASA, and the Center for Disease Control. Through the work of a USDA acarologist, mite researchers nationwide utilized LTSEM to image soft bodied forms of insects and mites with clarity never before seen. These investigators provided specimens not widely available for study for use in the testing and documentation of this microscopy method and greatly assisted the work in this field without expenditures to USDA.

Documentation:

Carta L. K., Erbe E. F., Pooley. C., Murphy C. and Wergin W.P., 2003. Chemical fixation/ambient temperature SEM vs cryofixation/low temperature SEM for taxonomic studies of nematodes. Scanning, Vol. 25, (2) pp.54-56 March/April.

Bergin, W.P., E.F. Ere and R. Ochoa. 2001. Versatile and inexpensive specimen holder for high angle azimuth rotation in a low temperature scanning electron microscope. Proc. Micros. Soc. of Am., Microsc. and Microanalysis 7, pp 718-19.

Orion, D., G.Kritzman, SLF. Meyer, E.F. Erbe and D.J. Chitwood.  2001. A role of the gelatinous matrix in the resistance of root-knot nematode (Meloidogyne spp.) eggs to microorganisms. J. of Nematology 33(4):203-207.

Pena, J.E., R. Ochoa, E.F. Erbe. 2002. Polyphagotarsonemus latus (Acari: Tarsonemidae) Research status on Citrus. Proc. Int. Soc. Citriculture 2000. pp. 754-759.

Wergin, W. P., R. W. Yaklich, L. K. Carta, E. F. Erbe and C. A. Murphy. 2000. Effect of an Ice-Nucleating Activity Agent on Subzero Survival of Nematode Juveniles.   Journal of Nematology 32(2):198-204.

Discovery Area 4:  Contributions to the Development of Regulations Issued by Governmental Agencies

Molecular and Morphological Systematics of Plant-Pathogenic Nematodes

Accomplishment:  The wheat seed gall nematode, a nematode of international quarantine importance (not reported in the United States since 1975) was not detected in any wheat samples sent from numerous U.S. locations. However, ARS scientists did discover another devastating seed gall nematode, Afrina wevelli, in love grass seeds from South Africa.

Impact: Confirming the absence of the wheat seed gall nematode contributed to the resumption of the $80 million annual wheat trade with Brazil, for which ARS scientists were awarded an Honorable Mention by the Federal Laboratory Consortium for Technology Transfer in 2002. APHIS regulations were implemented against another devastating seed gall nematode, Afrina wevelli, in love grass seeds from South Africa.

Additional information:  ARS hosted a visit by Brazilian administrators and scientists to jointly inspect and certify that U.S. wheat is seed gall nematode-free; a partnership between ARS, APHIS, and FAS facilitated this visit.

Exotic viruses of sweet potatoes

Accomplishment:  Scientists may import small quantities of sweet potato germplasm if it undergoes quarantine and indexing for exotic pathogens, primarily viruses and phytoplasmas. An ARS service program is responsible for conducting the quarantine and testing of sweet potato germplasm. A new procedure developed by ARS was shown to be as efficient as the previous, longer quarantine procedure in detecting exotic pathogens that could severely damage U.S. sweet potato production. The new quarantine and indexing program can be completed in only one growing season (6-9 months).

Impact:  APHIS approved this modified quarantine and indexing program in 2004, which reduced the quarantine period by more than half. Consequently, the shortened quarantine period for sweet potato will deliver new germplasm to breeders and other recipients in half the time. This germplasm will be used to introduce disease resistance and desirable horticultural traits into new varieties for U.S. markets. This protocol may also serve as a model for sweet potato certification and virus indexing programs.  

Biological Control Component

Introduction:  Biological control, although environmentally desirable, is often unreliable and difficult to implement.  Knowledge of the identity and mode of action of organisms that interfere with pathogens is needed to develop more effective biological control strategies for reduction of crop losses.  Knowledge of the ecology of biological control agents, how they affect and are affected by other organisms, and how biological control is influenced by factors such as weather, soil type, conventional fungicide application, and crop variety is needed to make this strategy more effective and reliable.  Additionally, information on how fermentation protocols influence biological control agent production, stability, and amenability to formulation must be obtained if the goal of developing more effective biological control products is to be achieved.  Additional knowledge is required to identify physical and biologically-based approaches that enhance the performance of biocontrol agents and/or induce host resistance.

Discovery Area 1: Biological Control Foliage and Fruit Diseases

Biological control for cacao and other alternative crops to illicit narcotic crops

Background:  Cacao, vegetables, and other crops, which may be alternatives to illicit narcotic crops, are susceptible to many fungal diseases. Among these diseases are those caused by Phytophthora spp. (damping-off, root, crown and pod rots, and blights) on cacao and many other crops, and those diseases specifically on cacao caused by Crinipellis perniciosa (witches' broom and pod rot) and Moniliophthora rorei (frosty pod rot). Successful cultivation of alternative crops is dependent on managing these diseases using integrated disease management strategies, preferably in a sustainable crop production system. Biological control, using antagonistic and parasitic microorganisms indigenous to the areas where the crops are grown, offers a promising approach to disease control, especially in environmentally sensitive areas such as the tropics. Extensive research is being carried out to identify, characterize, and develop biological control and delivery strategies using microorganisms that improve crop health. Mechanisms used by microbes to influence resistance of the crops to plant pathogens are being evaluated and characterized. A systems approach to disease control incorporating biocontrol technology will enable the development of sustainable crop production strategies that will allow management of disease outbreaks and reduction in crop disease losses.

Accomplishment:  An epidemiological weather station was designed, constructed and installed in Bahia, Brazil, for the study of beneficial and pathogenic organisms on cacao.  This effort included the design, construction and installation of a research platform for conducting epidemiological studies through the biosphere of the cacao tree from the soil to the upper canopy.  This equipment has resulted in the completion of experiments to evaluate the behavior of beneficial fungi in controlling witches broom at different parts of the tree in relationship to microclimates.

Impact:  Biologists have been studying the performance of commercial biocontrol applications for the control of Witches’ Broom in cacao for many years.  The data collected has been very variable from season to season and year to year.  The weather station that was developed has already yielded results explaining variability of biocontrol performance due to environmental fluctuation over time and within the forest canopy.  This data will allow the optimization of formulation and application techniques, as well as selection of the best adapted biocontrol isolates for use in cacao.

Additional Information:  Research was carried out through a SCA with Pennsylvania State University.   

Biologically-based management of fire blight of apple and pear trees

Background:  Fire blight is a serious disease of apple and pear trees caused by the bacterium, Erwinia amylovora.  The primary infection site is the blossom, where E. amylovora becomes established on the stigma during warm weather and can migrate in free moisture and eventually invade through nectary openings.  The importance of the disease has increased in the Pacific Northwest due to bacterial resistance to the antibiotic, streptomycin, and the planting of more susceptible apple varieties and rootstocks.  Suppression of E. amylovora with beneficial bacteria is a relatively new alternative or complementary control strategy.  Novel laboratory screening techniques involving the use of detached crab apple flowers led to the discovery of Pantoea agglomerans strain E325, which was exceptionally effective in suppressing E. amylovora on flower stigmas.  A U.S. Patent for the use of strain E325 for fire blight management was issued in July 1999. 

Accomplishment:  At the beginning of 2000, a patent license agreement and CRADA (extending to August 31, 2004) with Northwest Agricultural Products (NAP) were in place to commercially develop P. agglomerans strain E325 for controlling fire blight of apple and pear.  In addition to the wild type E325 strain, NAP was provided with derivative antibiotic-resistant strains to monitor populations in the orchard and ensure compatibility with an antibiotic used commercially.  This work contributed to the development of a suitable fermentation medium and product formulation by sharing information obtained through laboratory experience, determining the quantity and purity of bacterial cells in NAP samples, and conducting efficacy trials with detached crab apple flowers in the laboratory and on apple trees in the field.  Under the name “Bloomtime Biological,” the product was field tested in different fruit-producing regions and shown to exceed the efficacy of BlightBan A506, the only biological product currently available for fire blight in the United States.  NAP anticipates full EPA registration for the new product sometime in 2005.

Impact:  Pantoea agglomerans strain E325, marketed as Bloomtime Biological, will serve as an effective tool in the management of fire blight of apple and pear in the United States.  It will complement other control strategies and significantly reduce tree and fruit losses attributed to this disease.  Efforts to understand the mechanism of strain E325 will possibly lead to advancements in biological control of fire blight with this or related organisms.    

Additional Information:  Small grants were received from the Washington Tree Fruit Research Commission to work on biological control of fire blight, and the Winter Pear Control Committee and the Northwest Horticultural Council to conduct research addressing fire blight phytosanitary issues affecting the export of U.S. pears.

Documentation:

Pusey, P.L. 2000. The role of water in epiphytic colonization and infection of pomaceous flowers by Erwinia amylovora. Phytopathology 90:1352-1357.

Pusey, P.L. 2002. Biological control agents for fire blight of apple compared under conditions limiting natural dispersal. Plant Dis. 86:639-644.

Pusey, P.L, and Curry, E.A. 2004. Temperature and pomaceous flower age related to colonization by Erwinia amylovora and antagonists. Phytopathlogy 94:901-911.

Discovery Area 2: Biological Control of Soilborne Pathogens

The microbial and molecular basis of take-all decline

Background:  In the United States, wheat is grown on 75-80 million acres of land and barley is grown on 10 million acres. Root diseases, including take-all, Rhizoctonia, Pythium, and common root rots, and Fusarium foot rot, caused by soilborne fungal pathogens are major production limiting factors in cereal-based cropping systems. Resistance to foliar diseases is common in plants but crop species including wheat and barley lack resistance to many of the most widespread soilborne pathogens.  Thus, plants have adopted a strategy of selectively stimulating and supporting populations of antagonistic microorganisms in the rhizosphere environment as the first line of defense against attack by soilborne pathogens. The best example of natural disease suppressiveness is take-all decline (TAD), which is the spontaneous remission in the incidence and severity of take-all during wheat and barley monoculture, following a severe outbreak of the disease. TAD is a global phenomenon and a 1997 survey revealed that many wheat farmers across the United States utilize wheat monoculture to control take-all.  Buildup of antagonistic microorganisms in the rhizosphere environment during monoculture was thought to be responsible for TAD, but the exact microbial and molecular basis of the suppressiveness remained a mystery.

Accomplishment:  It was demonstrated that TAD results from the buildup in the rhizosphere environment of fluorescent Pseudomonas spp., producing the antibiotic 2,4-diacetylphloroglucinol (DAPG). Genetic probes and primers specific for phlD, a key gene in the DAPG biosynthetic locus, in combination with colony hybridization and PCR, were used to demonstrate that DAPG producers established threshold population densities of >10⁵  DAPG producers/gram of root (required for disease suppression) in TAD soils but remain below the threshold population on wheat grown in non-suppressive soils. Transfer of TAD soil to non-TAD (conducive) soil established threshold densities of DAPG producers and suppressiveness, and reduction of DAPG producers below the threshold eliminated suppressiveness. DAPG was isolated from roots grown in TAD but not conducive soils.  It was shown that enrichment of DAPG producers by wheat monoculture was a common phenomenon throughout the United States, including in Fargo, North Dakota on the North Dakota State University continuous spring wheat plot No. 2, established in the 1882 by W. M. Hayes and listed in the National Register of Historic Places.  DAPG-producing fluorescent Pseudomonas spp. were shown to be responsible for TAD in Dutch soils.  Repetitive-sequence based (rep) PCR and RFLP and sequence analysis of phlD identified 18 distinct genotypes (A–Q & T) of DAPG producers, however, in Washington TAD soils D-genotype isolates are primarily responsible for disease suppression because of their unique affinity for wheat roots.  Introduction of very small doses of D-genotype isolates into conducive soils duplicates TAD and the bacteria remain at threshold densities in the soil as long as wheat is grown. 

Impact: TAD soils were described over 60 years ago and are used throughout the world to control take-all, one of the most important root diseases of wheat. These studies are the first to describe the molecular basis of take-all decline or any suppressive soil, and ended 50 years of debate about the biological basis of TAD. These findings have rejuvenated research on suppressive soils by plant pathologists and microbiologists using molecular biology approaches, and have resulted in greater awareness of the potential of suppressive soils to control soilborne pathogens in sustainable farming systems. As a result of these studies, DAPG producers are now recognized as playing key roles in plant defense of other crop species grown in monoculture.  This technology has been patented and D-genotype strains are under test for commercial development.

Additional Information:  The research was conducted in collaboration with the Department of Plant Sciences, Laboratory of Phytopathology, Wageningen University, Wageningen, The Netherlands.  Competitive grants supported research that contributed to this accomplishment and NRI.

Documentation:

McSpadden Gardener, B. B., Schroeder, K. L., Kalloger, S. E., Raaijmakers, J. M., Thomashow, L. S., and Weller, D. M. 2000. Genotypic and phenotypic diversity of phlD-containing Pseudomonas isolated from the rhizosphere of wheat. Applied and Environmental Microbiology. V. 66, p. 1939-1946. 

Raaijmakers, J. M., and Weller, D. M. 2001. Exploiting genotypic diversity of 2,4-diacetylphloroglucinol-producing Pseudomonas spp.: Characterization of superior root-colonizing P. fluorescens strain Q8r1-96. Applied and Environmental Microbiology. V. 67, p. 2545-2554.

Weller, D. M., Raaijmakers, J. M., McSpadden Gardener, B. B., and Thomashow, L. S. 2002. Microbial populations responsible for specific suppressiveness to plant pathogens. Annual Review of Phytopathology. V. 40, p. 309-348.

de Souza, J. T., Weller, D. M., and Raaijmakers, J. M.  2002. Frequency, diversity, and activity of 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp. in Dutch take-all decline soils. Phytopathology. V. 93, p. 54-63.

Landa, B. B., Mavrodi, D. M., Thomshow, L. S., and Weller, D. M. 2003. Interactions between strains of 2,4-diacetylphloroglucingo-producing Pseudomonas fluorescens in the rhizosphere of wheat. Phytopathology. V. 93, p. 982-994.

Biological control of soilborne pathogens of potato in New England cropping systems

Background:  Agricultural production in the New England Region has seriously declined in recent years. Within the past decade alone, farmland in New England has decreased by 393,000 acres and potato production, once a major rotation crop in New England agriculture, has seriously declined because of competition from other potato growing regions in the United States and diseases.  One of the most important diseases in the New England region is Late Blight (Phytophthora infestans), which causes significant losses to the potato and tomato industries every year.  Sustainable cropping systems and disease management practices are needed that rely on biological control and other natural methods of disease control.

Accomplishment:  Potatoes suffer from numerous plant diseases, and billions of dollars are spent annually in the United States to control these diseases. Sustainable methods of disease control were evaluated that include rotations, green manures, cover crops, and organic amendments, along with biocontrol agents, microbial inoculants, biostimulants, and novel biocontrol organisms.  Commercially-available biological control organisms such as Trichoderma virens and Bacillus subtilis reduced Rhizoctonia stem canker by 37-75% and black scurf by 11-20%, while increasing potato yield by 15-20%.  These practices/amendments have significant potential for improving disease control and reducing pesticide inputs.  

Impact:  The development and transfer of sustainable biocontrol technologies to New England growers has helped improve agricultural viability and rural economic vitality in the Northeast.

Additional Information:  The Maine Potato Board has contributed funding to evaluate potential controls for powdery scab.  This has facilitated performing on-farm research that has identified the potential of Brassica crops for controlling powdery scab and other soil-born diseases. 

Documentation:

Larkin, R.P. 2003. Characterization of soil microbial communities under different potato cropping systems by microbial population dynamics, substrate utilization, and fatty acid profiles. Soil Biology & Biochemistry 35:1451-1466.

Biology and management of soilborne diseases and beneficial soil and root-inhabiting microorganisms in nursery and other horticultural crops

Background:  Soilborne diseases of nursery and other horticultural crops cause serious losses to growers in the United States and throughout the world.  Control or management strategies are limited at present, with growers relying heavily on chemical agents to eliminate or suppress these diseases.  One such approach is preplant fumigation of the soil in nurseries or other plantings to eliminate or greatly reduce pathogen populations in the soil, but that approach has significant risks to workers and to the environment.  This  research has been largely focused on finding alternative approaches to controlling such pathogens, especially biological and cultural methods.  This work has emphasized the roles played by beneficial microorganisms in the rhizosphere soil of crop plants, especially rhizobacteria and mycorrhizal fungi and has emphasized those cultural practices that most affect the establishment and function of mycorrhizal fungi and bacterial biocontrol agents within the production system. 

Accomplishments:  Mycorrhizal fungi are beneficial fungi that form a symbiotic relationship with most plants on Earth.  Much of the nursery industry employs soilless plant growth media in most or all of the production systems.  Mycorrhizal fungi are inhibited by many of the cultural practices commonly used, especially heavy use of fertilizers and pesticides.  In a series of studies many of those practices, especially the media components and the application of fertilizers have been addressed.  The works has shown that formation and function of mycorrhizae in soilless media can be affected adversely by some peat mosses used, by some forms of organic or inorganic fertilizers (especially with high P content), and by most composts.  These studies were reported in a series of papers in the horticultural journals.  Currently, these studies have shown that formation of arbuscular mycorrhizae stimulates an increase in population of antagonistic rhizobacteria in rhizosphere soil, thus indicating a role for these fungal symbionts in the biological control of soilborne fungal pathogens.

Impact:  Understanding the factors in usual production practices of nursery crops that might favor the establishment of beneficial microbes, such as mycorrhizal fungi and antagonistic rhizobacteria is key to modifying practices to accommodate these beneficial organisms as a means of suppressing soilborne pathogens.  Growers now have some guidance as to how to favor beneficials as a means of controlling soilborne pathogens rather than relying strictly on the application of chemical agents.

Documentation:

Linderman, R. G., Davis, E. A. 2004. Evaluation of commercial inorganic and organic fertilizer effects on arbuscular mycorrhizae formed by Glomus intraradices. HortTechnology 14(2): p. 196-202.

Linderman, R. G., Davis, E. A. 2005. Comparative plant susceptibility and Phytophthora species’ virulence on detached nursery crop leaves.  Submitted to journal, in revision.

Brodhagen, M., Henkels, M. D., and Loper, J. E. 2004.  Positive autoregulation of the antibiotic pyoluteorin in the biological control organism Pseudomonas fluorescens Pf-5.  Appl. Environ. Microbiol. 70:1758-1766.

Stockwell, V. O., Johnson, K. B., and Loper, J. E. 2002.  Antibiosis contributes to biological control of fire blight by Pantoea agglomerans strain Eh252 in the field.  Phytopathology 92: 1202-1209.

Anderson, L.M., Stockwell, V.O., and Loper, J.E. 2004.  An extracellular protease of Pseudomonas fluorescens A506 inactivates antibiotics of Pantoea agglomerans.  Phytopathology 94:1228-1234.

Pinkerton, J. N., Schreiner, R. P., Ivors, K. L., Vasconcelos, M. C. 2004. Effects of Mesocriconema xenoplax on Vitis vinifera and associated mycorrhizal fungi. Journal of Nematology 36:193-201.

Critical assessment of compost teas for disease control

Background:  There has been increased popular interest in the utilization of compost teas to help improve plant health.  However, there are few critical evaluations of their utility for disease control despite numerous popular claims.  In addition, within the popular press there were numerous claims that were contradictory to the current scientific evidence.  We endeavored to systematically evaluate the utility of compost teas and numerous tea production methods for control of plant diseases.

Accomplishment:  It was demonstrated that popular press claims that aerobically produced compost teas (requiring expensive equipment) were superior to nonaerated (i.e., anaerobic) compost teas in controlling foliar and soilborne diseases were misleading and that nonaerated compost teas did not cause phytotoxicity as is commonly claimed.  It was also established that compost source was more important than the tea production method in obtaining disease suppressive compost tea.  The consistency of disease control was increased through the addition of spreaders and stickers that increased the distribution of microbes on the leaf surface.                                                 

Impact:  This research generated critical knowledge and addressed numerous misconceptions so that practitioners choosing compost teas as a disease management tool now have a solid foundation from which to make decisions.  This research is particularly beneficial to organic growers since they have few options for disease management.  

Additional Information:  The research on Compost Tea was conducted in collaboration with Oregon State University.  Outside funding came from Oregon Department of Agriculture and the Oregon Nurserymen Association.

Documentation:

Scheuerell, S.J. Sullivan, D. and Mahaffee, W.F. 2005. Suppression of Seedling Damping-Off Caused by Pythium ultimum, Pythium irregulare, and Rhizoctonia solani in Compost Amended Container Media.  Phytopathology 95:306-315.

Scheuerell, S.J. and Mahaffee, W.F. 2004. Compost Tea as a Container Media Drench for Suppressing Seedling Damping-Off Caused by Pythium ultimum. Phytopathology 94:1156-1163

Antagonistic microbes for management of plant-parasitic nematodes

Background:  Plant-parasitic nematodes are microscopic soil worms that reduce the yield and quality of many crops. Nematodes cause nearly ten billion dollars in crop losses per year in the United States and nearly 100 billion dollars globally. For some crops, such as soybeans, nematodes are the most important pest or pathogen. However, many of the conventional nematicides that were used to control plant-parasitic nematodes have been shown to contribute to groundwater contamination and to be hazardous to the health of humans and animals, and have therefore been banned or restricted in use. It is particularly notable that the restrictions on methyl bromide application will remove the most widely used nematicide from agricultural use. Consequently, new, effective and environmentally safe tools for managing nematodes are urgently needed.  ARS is developing nematode management systems based upon three different strategies: 1) determining the usefulness of antagonistic fungi and bacteria (alone and in combination with other organisms or practices), 2) identifying natural products that adversely affect nematodes, and 3) applying soil amendments to reduce nematode crop losses.

Accomplishment:  (1) Nematode-Antagonistic Microbes.  Biocontrol agents that are active against multiple pathogens or pests are more likely to be cost-effective in commercial formulations than organisms that are only active against a single target.  ARS Scientists and collaborators tested fungi and bacteria for production of compounds toxic to root-knot nematodes (RKN; Meloidogyne spp.), and for ability to act as biocontrol agents against RKN populations on pepper and cucurbit roots.  Many of the tested microbes had already been shown to be active against plant-pathogenic fungi.  Two of the tested microbes, the fungus Trichoderma virens and the bacterium Burkholderia cepacia, were isolates of species that are sold commercially for management of soilborne pathogens.  However, the ability of strains in the ARS collection to control nematodes was poorly known, and research demonstrated that the tested strains of Burkholderia and Trichoderma could suppress nematode populations on bell pepper.  

Impact:  (1) Nematode-Antagonistic Microbes.  This research contributed to the establishment of a collaborative agreement entitled "Development of Biological Controls for Suppression of Select Soilborne Diseases of Cucumber and Other Greenhouse‑grown Vegetables" with a private sector firm; a ARS scientist was involved in the work on this project. The microbes that were identified as active against nematodes could potentially be applied by growers in an integrated pest management system for reduction of certain plant diseases caused by fungi and nematodes.

Accomplishment:  (2) Natural products.  One potential means of managing plant-parasitic nematodes is by soil application of natural products as alternatives to fumigant and non-fumigant nematicides.  To obtain active natural products for management of soybean cyst nematode (SCN) and other plant-parasitic nematodes, fungi were isolated from SCN on soybean plants in China, the geographic location where SCN and its host plant (soybean) originated.  ARS scientists and collaborators isolated the fungi, imported the isolates to the United States, and identified the fungal isolates.  The imported fungi (254 isolates) were assayed for production of compounds that increased or reduced nematode egg hatch, or that affected mobility of hatched juvenile nematodes; assays were conducted with SCN and RKN.  The active natural products were isolated and identified from two fungal species, and one compound was chemically synthesized. 

Impact:  (2) Natural Products.  Scientists in the United States now have many fungi from China for evaluation in biocontrol programs; this is particularly important because this is the only research being conducted on imported exotic microbes for SCN management.  In addition, the developed assay successfully identified active natural compounds produced by nematode-associated fungi, and the natural products are being studied for biologically based management.  The research clearly confirmed that target nematode species must be used in assays for bioactive products, because compounds active against one nematode were not necessarily active against another.  This is significant because researchers frequently use bacterial-feeding nematodes (which are generally easier to culture than plant-parasites) when looking for active compounds to apply against plant-parasitic nematodes.  Natural products, when appropriately delivered to plants, may be used by growers to reduce crop losses caused by plant-parasitic nematodes.

Additional Information:  Researchers from ARS collaborated with scientists at the following locations: 1) Institute of Biological Control (Chinese Academy of Sciences, Beijing, China) for collecting fungi from China; 2) Fusarium Research Center (Pennsylvania State University) for fungal identifications; 3) University of MD for identifying and synthesizing fungal compounds toxic to nematodes.

Documentation:

Meyer, S.L.F., R.N. Huettel, X.-Z. Liu, R.A. Humber, J. Juba, and J. Nitao.  2004.  Activity of fungal culture filtrates against soybean cyst nematode and root-knot nematode egg hatch and juvenile mobility.  Nematology 6:23-32.

Zasada, I.A. and M. Tenuta.  2004.  Chemical-mediated toxicity of N-Viro Soil to Heterodera glycines and Meloidogyne incognita.  Journal of Nematology 36:297-302,

Chitwood, D.J.  2002.  Phytochemical based strategies for nematode control.  Annual Review of Phytopathology 40:221‑249.

Nitao, J.K., S.L.F. Meyer, W.F. Schmidt, J.C. Fettinger, and D.J. Chitwood.  2001.  Nematode-antagonistic trichothecenes from Fusarium equiseti.  Journal of Chemical Ecology 27:859-869. 

Meyer, S.L.F., S.I. Massoud, D.J. Chitwood, and D.P. Roberts.  2000.  Evaluation of Trichoderma virens and Burkholderia cepacia for antagonistic activity against root-knot nematode, Meloidogyne incognita.  Nematology 2:871-879.

Discovery Area 3: Biocontrol of Postharvest Diseases

Microbial production and formulation technologies for biocontrol of postharvest diseases of potato

Background:  Eighty percent of pathogen strains causing Fusarium dry rot of stored potato tubers are now genetically resistant to thiabendazole, the only fungicide registered for postharvest use.  Pseudomonads and other Gram-negative bacterial strains capable of dry rot suppression were identified in soil samples from fields with low disease incidence and ranked on the basis of growth kinetics in liquid culture and subsequent efficacy when delivered to wounded potatoes challenged with pathogen.  Subsequent studies indicated that all of the dry rot antagonists produced at least one, and often multiple antifungal compounds active against the pathogen Fusarium sambucinum.  Plant growth regulating compounds are also known to be produced by many pseudomonads.  Liquid culture nutrition was shown to influence the accumulations of bioactive metabolites and impact biocontrol of dry rot as well as other potato storage diseases and sprouting.  Therefore studies elucidating biocontrol agent metabolite bioactivities, dependence of metabolism on culture conditions, and target pest range are critical to enhancing commercial potential. 

Accomplishment:  Biological control strains and production/formulation protocols for biological control treatments capable of protecting postharvest potatoes in storage from dry rot, sprouting and late blight was discovered. Discoveries leading to this accomplishment included: the finding that several of our top dry rot antagonists suppressed sprouting in the laboratory and then three strains were able to inhibit sprouting in pilot trials for 4-5 months at a level similar to the chemical inhibitor 16 ppm CIPC thermal fog; observations that biocontrol success by any given bacterial strain was dependent on production culture nutrients, cultivar and pathogen strain; the discovery that the plant growth hormone indoleacetic acid (IAA) had antifungal activity against dry rot and that cultures of two bacteria strains could accumulate IAA in concentrations sufficient to cause sprout suppression when provided optimal nutrients in culture; the discovery that applying mixtures of top biocontrol strains was not detrimental and often enhanced efficacy and consistency of control from trial to trial; the discovery that bacterial antagonists could retain 60-90% of original population after 24 hours in the presence of one quarter strength CIPC sprout inhibitor and that the CIPC application rate needed for sprout control could be reduced by 75 to 90% if used in combination with the biological control strains; the result that late blight suppressiveness was indicated for several of our top dry rot suppressive strains in a laboratory screening of strains x production media x formulation; and the finding that top dry rot antagonistic bacteria were able to significantly suppress late blight infections by 20 to 90% the first year and 35 to 91% in the second season of pilot testing.  

Impact:  This accomplishment allows biocontrol of multiple potato storage problems including dry rot, late blight, and sprouting of stored potatoes with a single biocontrol treatment sprayed to potatoes as they enter storage, much like the traditional application of the now ineffective chemical TBZ for control of fungal diseases of tubers in storage.  The development of commercial biocontrol agents to address these postharvest storage problems could save U.S. potato growers $100-500 million per year in crop losses due to dry rot and late blight diseases alone.  Sprout control, required for 50% of the potato crop, is an added benefit of our disease suppressive biocontrol agents, and may allow reduced use of CIPC which faces tightening EPA regulation even though it is the only sprout inhibitor approved for postharvest use in the United States.  Multiple strains were patented as biological control agents against dry rot [Slininger, Schisler, and Bothast (1996), US Patent 5,552,315; Schisler, Bothast, and Slininger (1998), US Patent 5,783,411] and, during this review period, sprouting [Slininger, Burkhead, Schisler, and Bothast (2000), U.S Patent 6,107,247].  

Additional Information:  Collaboration with researchers at Mercer University, Macon, Georgia, led to the isolation of a compound from a culture of dry rot antagonistic bacteria which stimulated sprout suppressive bioactivity in the presence of the bacteria and which also exhibited dry rot suppressive bioactivity.  The compound has now also been shown to reduce the development of late blight caused by Phytophthora infestans infection of stored potatoes.  A simple derivative of this natural product applied at relatively low dosage has exhibited the ability to nearly completely inhibit the growth of P. infestans on plates and to suppress the development of late blight symptoms in stored potatoes. 

A current Cooperative Research and Development Agreement with an agricultural company located in the Pacific Northwest is partially funding pilot testing of our biological agents for control of dry rot, sprouting and late blight and enabling the company to make a licensing decision regarding our technology.  The University of Idaho Kimberly Research Station has provided expert assistance in designing potato storage experiments and has been the site of our annual pilot experiments to test new biocontrol agent formulations and activities discovered in laboratory experiments.   These collaborations have given us invaluable exposure to real world problems and practices in the potato industry, as well as large-scale fermentation techniques, traditional formulation techniques, marketing considerations, and commercialization requirements that must be met in order to realize a biocontrol product for use on postharvest potatoes.

Documentation:

Schisler, D.A., Slininger, P.J., Hanson, L.E., Loria, R.  2000.  Potato cultivar, pathogen isolate, and antagonist cultivation medium influence the efficacy and ranking of biological control strains of Fusarium dry rot.  Biocontrol Science and Technology 10:267-279.   

Slininger, P.J., Schisler, D.A., Burkhead, K.D., Bothast, R.J.  2003.  Postharvest biological control of potato sprouting by dry rot antagonistic bacteria.  Biocontrol Science and Technology 13(5):477-494.

Slininger, P.J., Behle, R.W., Jackson, M.A., Schisler, D.A.  2003.  Forum:  Discovery and development of biological agents to control crop pests.  Neotropical Entomology 32(2):183-195. 

Slininger, P.J., Schisler, D.A., Kleinkopf, G.  2004.   Biological control of post harvest late blight of potatoes in storage.  Phytopathology 94 (6):S96.  [2004 Poster Proceedings of The American Phytopathological Society,  July 31-August 4, Anaheim, CA (on compact disk)].

Slininger, P.J., Burkhead, K.D., and Schisler, D.A.   2004.  Antifungal and sprout regulatory bioactivities of phenylacetic acid, indole-3-acetic acid, and tyrosol isolated from the potato dry rot suppressive bacterium Enterobacter cloacae S11:T:07.  Journal of Industrial Microbiology and Biotechnology 31:517-524.

Emerging technologies to control postharvest diseases of citrus

Background:  Fresh fruit are attacked by fungal diseases that rot fruit after harvest. The fungal diseases that rot fruit after harvest cause losses of about 5% of California's $3 billion production. The control of the disease-causing fungi is important, and is usually accomplished using chemical fungicides and sanitizers, but issues of pest resistance to these chemicals, the dietary safety of their presence foods, and their impact on the environment has made the search for safer replacements important. Fungicides used to control these diseases interfere with the export of citrus fruit because some countries will not accept residues of the fungicides on the fruit. Three fungicides are approved for citrus fruit use in California, sodium ortho-phenyl phenate, imazalil, and thiabendazole. Sodium ortho-phenyl phenate is classified as a probable human carcinogen and imazalil as a possible human carcinogen by the U.S. Environmental Protection Agency.

Accomplishment:  Effective combinations of a biological control bacterium (Pantoea agglomerans) and postharvest thermal regimes were developed to control postharvest decay of citrus fruit. This technique reduces fungicide costs, decay losses by packers, and fungicide residues in citrus products purchased by consumers. In a series of experiments to establish optimum regimes, citrus fruit to which the bacterium had been applied were placed under warm, humid storage for up to 3 days at up to 44°C.

Impact:  This project investigated one strategy to manage and minimize postharvest decay losses of citrus fruit without fungicides, the integrated combination of citrus postharvest thermal treatment and a well-studied biological control antagonist. This approach is novel and not yet in commercial use, although pilot-scale demonstration experiments are now in progress in California and Spain.

Additional Information:  Supplemental funds for this work were contributed by the California Citrus Research Board. Much of this work was done in collaboration with a visiting scientist of the IRTA, University of Lerida, Lerida, Spain. The collaboration was funded entirely by grants from the Spanish citrus industry and Catalonian government.

Documentation:

Plaza, P., Usall, J., Smilanick, J. L., Lamarca, N. and Viñas, I.  2004. Combining Pantoea agglomerans (CPA-2) and curing treatments to control established infections of Penicillium digitatum on lemons. J. Food Protection V. 67 p. 781-786.

Plaza, P., Usall, J., Torres, R., Abadias, M., Smilanick, J. L., and Viñas, I.  2004. The use of sodium carbonate to improve curing treatments against green and blue moulds on citrus fruits. Pest Management Science V. 60 p. 815-821.

Smilanick, J. L., Sorenson, D., Mansour, M., Aieyabei, J.,  and Plaza, P. 2003. Impact of a brief postharvest hot water drench treatment on decay, fruit appearance, and microbe populations of California lemons and oranges. HortTechnology V. 13 p. 333-338.

Palou, L., Smilanick, J. L., Crisosto, C. H., Mansour, M., and Plaza, P. 2003. Ozone gas penetration and control of the sporulation of Penicillium digitatum and Penicillium italicum within commercial packages of oranges during cold storage.  Crop Protection V. 22 p. 1131-1134.

El-Ghaouth, A.; Smilanick, J. L.; Brown, G. E.; Ippolito, A.; Wilson, C. L. 2001. Control of decay of apple and citrus fruits in semi-commercial tests with Candida saitoana and 2-deoxy-D-glucose. Biological Control V. 20 p. 96-101. 2001.

Biological control of postharvest diseases of deciduous fruit trees

Background:  When taking into account the wide array of conditions present in packinghouse facilities (different cultivars, different levels of fruit maturity, different levels of inoculum pressure, etc.), the performance of postharvest biological control agents is generally more variable than for chemical fungicides. For example, as fruit mature, higher concentrations of an antagonist must be used to achieve the same level of decay control as on less mature fruits. Additionally, current postharvest biocontrol products are only protective and do not exhibit eradicant activity. Therefore, the efficacy and performance reliability for biocontrol must be increased. This is being accomplished through ARS studies on the mode of action of postharvest biocontrol agents, combining the use of biocontrol agents with natural compounds and/or heat treatments, and genetic engineering.

Accomplishments:  ARS scientists at Kearneysville, West Virginia, discovered and patented two biocontrol agents that were commercialized as the products, Aspire and Bio-Save, through a CRADA with industry. Subsequently, ARS scientists have found that the efficacy of yeast antagonists can be greatly enhanced by the addition of natural, bioactive compounds.  This technology has been patented and is being commercialized via a CRADA with industry as a “bioactive coating@ that can serve as an effective alternative to synthetic fungicides for the control of postharvest diseases of pome and citrus fruit.

A laboratory screen was devised that allowed the discovery of a number of natural volatile fragrance/flavor compounds and anti-microbial compounds to two major food contaminating bacteria (Escherichia coli and Pseudomonas marginalis) and two mold fungi (Botrytis cinerea and Penicillium expansum). When these natural anti-microbial compounds are released in the headspace of plastic bags containing fruit and vegetables they extend the shelf life of these commodities.  A new “active” plastic food storage bag design was invented that responds to moisture released from commodities stored in it by releasing carbon dioxide. The increased carbon dioxide concentration in the bag reduces microbial growth and extends the shelf life of the stored commodity.  Natural anti-microbial volatile compounds can also be used at lower concentrations when they are combined with this “active” carbon dioxide generating technology.

In collaboration with USDA-ARS scientists at Beltsville, Maryland a new strategy for integrated fruit decay control was developed.  This strategy combines heat treatments with hot air (4d at 38C), and sodium bicarbonate with biological control by bacterial and heat tolerant yeast antagonists. Combination of treatments also resulted in better control than either treatment alone. These treatments complemented each other in that heat provided eradicative activity up to 24 h after inoculation with P. expansum, and biocontrol agents provided residual activity. The heat or sodium bicarbonate treatments alone can not provide adequate decay control by themselves, but biological control combined with those methods provided decay control that is broader in the spectrum of activity and more efficient than the individual treatments.  A heat tolerant yeast, Metschnikowia pulcherrima, isolated from wounded apple in an unmanaged orchard was used.  This yeast is a common inhabitant of fruit including apple and grape, is commonly found in apple cider and is an integral part in the wine-making process. It was found that the use of 1-MCP on harvested fruit to inhibit maturation can predispose fruit to decay, but the alternatives to fungicides are capable of preventing this increase in decay.

A phage treatment was found to be very effective in reducing populations of Listeria moncytogenes on fresh cut melon. The most effective were cocktail of phages at concentrations around 10⁸ PFU/ml and when the application was up to 1h before contamination of the fruit. This implies that under commercial conditions the phage would have to be applied at the time of fruit cutting or shortly thereafter. It was found that a bacterium, that commonly occurs in fruit juices, and a biocontrol yeast were very effective in controlling L. monocytogenes and Salmonella Poona on fresh cut apples.  Use of the phage cocktail in combination with these biocontrol agents for control of L. monocytogenes and Salmonella Poona on fresh cut fruits is currently being explored.

Impact:   The development of potential products described above with increased efficacy and the ability to eradicate pre-existing infections will provide a new approach to postharvest disease control.  Additionally, the research has extended the potential application of biological approaches to disease control to the consumer.  While still in the development phase, industry has shown a great interest in using this technology in plastic storage bags.  This research demonstrated that the biological control of postharvest diseases can be easily integrated with other methods with additive and synergistic effects, which also broaden the spectrum of activity (eradicative effect of heat) and further advanced the goal toward eliminating fungicide treatment on fruits after harvest.  The work provided recommendations for the future integration of biocontrol with the other treatments.

Additional Information:  Research on the development of bioactive coatings was supported through a CRADA with industry over a 5 year period and the work on active packaging was also supported through a CRADA with industry.  Fundamental research on the role of lytic peptides on biocontrol efficacy and genetic engineering was supported by the Bi-National Agricultural Research and Development Fund  (BARD).

Documentation:

Bassett, C.L., and Janisiewicz, W.J. 2003. Electroporation and stable maintenance of plasmid DNAs in a biocontrol strain of Pseudomonas syringae. Biotechnology Letters 25:199-203.

Janisiewicz, W. J., Leverentz, B, Conway, W. S., Saftner, R. A., Reed, A. N., and Camp, M. J. 2003. Control of bitter rot and blue mold of apples by integrating heat and antagonist treatments on 1-mcp treated fruit stored under controlled atmosphere conditions. Postharvest Biology and Technology.  29: 129-143.

Norelli, J. L., and Miller, S. L. 2004. Effect of prohexadione-calcium dose level on shoot growth and fire blight in young apple trees. Plant Disease 88:1099-1106.

Wilson, C. L. and Peach, P.A. Docket No. 0013.03. “Compartmentalized Plastic Containers for the Segregation and Delivery of Compounds/Carriers in Active Packaging.” (11/12/2002).

Wisniewski, M., Bassett, C.L., Artlip, T., Webb, R., Janisiewicz, W., Norelli, J., Goldway, M., and Droby, S. 2003. Characterization of a defensin in bark and fruit tissues of peach and antimicrobial activity of a recombinant defensin in the yeast, Pichia guilliermondii. Physiologia Plantarum. 119: 563-572.

Discovery Area 4: Culturing, Formulation and Application Technologies for Biocontrol Agents

Culture and formulation technologies and protocols to develop more effective biocontrol agents

Background:  Thousands of potential microbial biocontrol agents have been discovered but fewer than 100 (40 antagonists of plant diseases) have reached the commercial production stage.  A major obstacle to biocontrol agent commercialization has been the lack of basic knowledge and experience with methodologies, techniques and tools for manufacturing, storage and delivery of cost effective products.

Accomplishment:  We have discovered numerous culture and formulation protocols influencing the abilities of biocontrol strains to tolerate stress factors encountered during production and formulation. Elements of this accomplishment included: development of a microplate-based high throughput screening technique to rapidly identify optimal cultivation, formulation, drying, and storage protocols that yield active biocontrol agents; identification of proline as an amino acid important to metabolite regulation and enhanced production of viable pseudomonads resistant to toxic culture endproducts;  identification of di- and tri-saccharide sugars such as lactose, turanose and melezitose as stabilizers during rapid drying of gram-negative bacteria; identification of relative humidity and sugar content requirements for long term survival of pseudomonads in dry storage; discovery of  the optimal carbon to nitrogen ratio and carbon loading for cell cultivation and also a unique cold shock protocol to finish the cultivation and induce enhanced dry storage survival of a wheat scab suppressive yeast.

Impact:  These discoveries are key to the commercial feasibility of applying beneficial microbial agents for suppressing take-all and Fusarium head blight of wheat as well as dry rot, late blight and sprouting of postharvest potatoes in storage.   The development of commercial biocontrol agents to address these field and storage problems could save U.S. wheat growers up to $1.0 billion per year and potato growers $100-500 million per year in crop losses due to fungal disease.  Sprout control, required for 50% of the potato crop, is an added benefit of our disease suppressive biocontrol agents, and may allow reduced use of CIPC which faces tightening EPA regulation even though it is the only sprout inhibitor approved for postharvest use in the United States.  Numerous invited presentations and publications of these results by ARS scientists have convinced many scientists of the critical importance of optimizing the fermentation environment to enhance the amenability of biomass products to stresses encountered during formulation and stabilization.

Additional Information:  Collaboration with a Florida-based company provided evidence that pseudomonads could survive with less than 50% loss of viable units on dry nutrient granules for several months at 25^oC and could be used to reduce the chemical fertilizer needs of turf establishment.  This discovery has lead to more controlled experiments to determine cell survival requirements and to design granule composition to control cell survival to make this technique more generally applicable to the drying and storage of gram-negative bacteria for use in many areas of biological control.

In preparation of development of a new DNA oligo microarray for Pseudomonas fluorescens Pf-5, a plant disease biocontrol agent, a set of quality controls using exogenous nucleic acids was generated.  The reliability and reproducibility of microarray data are critical for obtaining meaningful biological insights from genomic expression analysis.  Quality control of microarray experiments is an important element for data handling of microarray experiments.  These quality controls were developed in defined concentrations and are ready for incorporation into each RNA labeling reaction. Due to the uniqueness of these selected control genes, our quality controls can be used as universal controls for any microbial DNA oligo microarrays, as well as for a wide range of microarrays for plant and animals.  In collaboration with ARS scientists in Corvallis, Oregon a DNA oligo microarray of the recently sequenced Pseudomonas fluorescens Pf-5 genome has been developed and printed.  Microarray analysis of Pf-5 genome will be used to study stress tolerance mechanisms of pseudomonads and how they can be regulated and controlled via cell cultivation and formulation techniques. In recognition for this work, an ARS scientist has been invited to represent ARS as a member of the External RNA Control Consortium (organized and sponsored by the Biotechnology Division, National Institute of Standards and Technology) to establish universal control protocols for the collection of microarray data from diverse genomes and laboratories.

Documentation:

Slininger, P.J., Schisler, D.A.  2003. High throughput assay for optimizing microbial biological control agent production and delivery.  Phytopathology. 93(6):S79 [2003 Poster Proceedings of The American Phytopathological Society, August 9-13, Charlotte, NC (on compact disk)]. 

Schisler, D.A., Slininger, P.J., Behle, R.W., and Jackson, M.A.  2004.   Formulation of Bacillus spp. for biological control of plant diseases.  Phytopathology.  94(11):1267-1271.

Zhang, S., Schisler, D.A., Jackson, M.A., Boehm, M.J., Slininger, P.J.  2004.  USDA-ARS, Ohio State University cooperative research on biological control of Fusarium head blight 2: Cold temperature shock during production of Cryptococcus nodaensis OH 182.9 enhances cell survival after air-drying.  In:  Proceedings of the 2^nd International Symposium on Fusarium Head Blight Vol. 2, December 11-15, 2004, Orlando, FL  pp. 383-387.

Slininger, P.J., and Shea-Andersh, M.A.  2005.  Proline-based modulation of 2,4-diacetylphloroglucinol and viable cell yields in cultures of Pseudomonas fluorescens wild-type and over-producing strains.  Applied Microbiology and Biotechnology.  (In press, on line 2-18-05 at http://www.springerlink.com/index/10.1007/s00253-005-1907-4).

Zhang, S., Schisler, D.A., Boehm, M.J., and Slininger, P.J.  2005. Carbon-to-nitrogen ratio and carbon loading of production media influence freeze-drying survival and biocontrol efficacy of Cryptococcus nodaensis OH 182.9.  Phytopathology (accepted 2/8/05).

Screening, production and formulation technologies for biocontrol agents of Fusarium head blight of wheat

Background:  The development of effective microbial selection, biomass production and biomass formulation methodologies for microorganisms capable of reducing plant diseases represents a vital step in the advance of biocontrol products that will provide attractive solutions to several vexing problems inherent with traditional plant disease control strategies.  A serious impediment to bringing antagonists to the marketplace is the lack of adequate knowledge of the mass production and formulation technologies needed to produce cells that are tolerant to the stresses of large-scale cultivation, separation, processing (drying or dewatering biomass) and storage.  We devise commercial practice-oriented biocontrol agent selection strategies and elucidate the genetic and metabolic mechanisms that enhance microbial stress tolerance such that high yields of viable, effective cells with long shelf-lives can be produced.  In the specific case of our work in discovering and developing novel biocontrol agents active against Fusarium head blight of wheat, the new technologies developed will ultimately contribute to reducing estimated losses of at least $500 million per year attributed to this disease in the United States.

Accomplishment:  Novel microbial antagonists for reducing Fusarium head blight of wheat were discovered and new liquid culture protocols and formulations were developed for producing biomass with enhanced efficacy and shelf-life.  Screening microbial colonists isolated from wheat anthers for ability to utilize compounds such as tartaric acid or choline enhanced the recovery of strains capable of reducing Fusarium head blight in greenhouse and field trials.  Choline utilizing strains were isolated based on the observation that choline is found in higher relative concentrations in tissues of susceptible flowering wheat heads and enhances germ tube elongation of the pathogen.  Novel production techniques were developed that enabled the mass production (100 L fermentations) and processing of biomass of the yeast antagonist Cryptococcus flavescens (C. nodaensis nomen nudum) as a frozen biomass concentrate with an extended shelf-life.  We discovered that formulating biomass of C. flavescens with the previously unreported osmoprotectant melezitose significantly enhanced the stability of freeze-dried preparations of antagonist biomass.  Significant reduction of FHB symptoms were achieved when grains were treated at flowering in field tests conducted over multiple sites and years on soft red winter wheat, hard red spring wheat and durum wheat. 

Impact:  Technology transfer has been achieved via the issuance of two patents and one pending patent [Schisler, Khan and Boehm (2001), US Patent 6,312,940; Schisler, Khan and Boehm (2003), US Patent 6,562,337] regarding the novel biocontrol strains discovered as well as multiple peer reviewed and technology transfer publications.  A Trust Fund Cooperative Research Agreement was completed with the Dakota Growers Pasta Company to conduct a field demonstration of the efficacy of several of our patented antagonists against FHB on durum wheat.  Due to the demonstrated potential of our FHB biocontrol strain C. flavescens, the U.S. Wheat and Barley Scab Initiative funded cooperative field research that was conducted with 13 State Universities at approximately 16 field sites over two successive years.  Project results led to a 2.5 year cooperative international research project with Former Soviet Union (FSU) Bioweapons Researchers that was funded through the auspices of ARS’s Office of International Research Program.  ARS and FSU collaborators made multiple visits to each other’s laboratories, tested biocontrol strains in each others countries and published results.  Invited presentations of these results by CRIS scientists at numerous national and international meetings have influenced production and formulation research of scientists in related and unrelated fields.  Ultimately these results contribute to advancing biocontrol strains in general towards commercialization by solving previously intractable problems of production, formulation and stabilization of biomass that must be met before commercial product development can be reasonably anticipated.

Additional Information:  Demonstration of the feasibility of reducing FHB on durum wheat using ARS-discovered antagonists was made possible in part by ARS obtaining a two year grant from the Dakota Growers Pasta Company.  Additionally, ARS maintained a Specific Cooperative Agreement with The Ohio State University from 2000-2004.  This SCA was funded primarily by competitive funding that was obtained by ARS from the U.S. Wheat and Barley Scab Initiative.  Expertise in the initiation and maintenance of wheat field trials was provided by collaborators at The Ohio State University.

Documentation:

Schisler, D.A., Khan, N.I., Boehm, M.J., and Slininger, P.J.  2002.  Greenhouse and field evaluation of biological control of Fusarium head blight on durum wheat.  Plant Disease 86:1350-1356.

Schisler, D.A., Behle, R.W., Slininger, P.J., and Jackson, M.A.  2003.  Production and formulation of microbial products active against plant pests.  Proceedings of the II Moscow International Congress of Biotechnology:State of the art and prospects of development, P&I JSC “Maxima”, Moscow, Russia. p. 263.

Khan, N.I., Schisler, D.A., Boehm, M.J., Slininger, P.J., and Bothast, R.J. 2001. Selection and evaluation of microorganisms for biocontrol of Fusarium head blight of wheat incited by Gibberella zeae. Plant Dis. 85:1253-1258.

Schisler, D.A., Slininger, P.J., Behle, R.W., Zhang, S., Boehm, J.J., Lipps, P.E., and Palmquist, D.E.  2003.  USDA-ARS, Ohio State University cooperative research on biologically controlling Fusarium head blight 1: in vitro and field testing of the effect of UV protectants on FHB antagonists.  Proceedings of the 2003 National Fusarium Head Blight Forum, Kinko’s, Okemos, MI. pp. 105-108.

Khan, N.I., Schisler, D.A., Boehm, M.J., Lipps, P.E., and Slininger, P.J.  2004.  Field testing of antagonists of Fusarium head blight incited by Gibberella zeae.  Biological Control 29:245-255.

Discovery Area 5: Molecular Biology and Systematics of Biocontrol Agents

Total Genome sequencing of Pseudomonas fluorescens Pf-5

Background:  Pseudomonas fluorescens Pf-5 inhabits the root surfaces (rhizosphere) of many plants and functions as a biological control agent, suppressing a number of plant diseases caused by soilborne plant pathogens. Pf-5 produces a large spectrum of secondary metabolites, including several antibiotics. The selection of P. fluorescens Pf-5 as the subject for genomic sequencing was based upon the importance of Pf-5 as a biological control organism, its rhizosphere competence, the large spectrum of antibiotics and other secondary metabolites that it produces, and its status as model environmental strain for studies of gene regulation. Strain Pf-5 was described early in the history of biological control research (Howell and Stipanovic 1979, 1980), and it was the first biological control agent for which the chemical basis of disease suppression was known.

Accomplishment:  The genome of the biological control agent Pseudomonas fluorescens Pf-5 was completely sequenced, and this is the first biological control agent for plant disease whose sequence is known. The genome was sequenced at the Institute for Genomics Research, under the direction of Ian Paulsen, in collaboration with ARS scientists and researchers at the University of  Arizona and Rutgers University. The genomic sequence of Pf-5 highlights several important characteristics of the biological control agent, including its production of multiple antibiotics toxic to plant pathogens, its utilization of plant-produced nutrients, its capacity to utilize siderophores produced by a broad range of soil microorganisms, and the lack of genes required for pathogenicity.  Sequence data will be used to develop basic knowledge of biological control, with the purpose of improving the consistent efficacy of biological control in agriculture.

Impact:  Sequence data is posted on a publicly accessible website that is used worldwide for access to genomic sequence data. The availability of a genomic sequence for a biological control agent will now allow scientists to employ a range of post-genomics technologies to enhance knowledge of biological control.  The genome of Pf-5 can now be compared to the genome of related bacteria such as Pseudomonas aeruginosa and Pseudomonas syringae.  Comparative genomics will advance knowledge of traits involved specifically in pathogenesis, as well as those involved in ecological fitness on plant surfaces or in soil.

Additional Information:  Outside funding from the USDA-CSREES Microbial Genome Sequencing Program was essential for this accomplishment, as was collaboration with The Institute for Genomic Research, Rutgers University, and the University of Arizona was key.  

Improving Pseudomonas fluorescens strains for biocontrol of soilborne pathogens of wheat

Background: Root diseases caused by soilborne fungal pathogens are major production limiting factors in cereal-based cropping systems, particularly when several consecutive crops are planted and when crops are direct-seeded into their own stubble (no-till). Wheat and barley lack resistance to many of the most common root pathogens, and populations of antagonistic rhizobacteria provide the first line of defense. These biological control bacteria suppress pathogens by producing phenazines and phloroglucinol antibiotics on the roots, but disease suppression can fail due to poor root colonization and inadequate antibiotic production. The goal of this ARS research is to develop bacterial biological control agents that consistently and effectively control root diseases caused by major cereal root fungal pathogens.

Accomplishment:  Strains of Pseudomonas fluorescens with superior root colonization ability and enhanced antibiotic production were developed by using classical and molecular microbiological approaches. Genomic fingerprinting techniques identified 18 unique BOX-PCR genotypes (A-Q & T) among 2,4-diacetylphloroglucinol (DAPG)-producing fluorescent Pseudomonas spp. D-genotype strains, exemplified by P. fluorescens Q8r1-96, showed a unique ability to aggressively colonize and persist on the roots of wheat and are responsible for take-all decline in Washington State wheat fields. Introduction of the biosynthetic locus for the antibiotic phenazine-1-carboxylic acid stably into strain Q8r1-96 resulted in transgenic strains that retained DAPG production, constitutively produce phenazine antibiotics, retained superior root colonizing ability, and were effective at lower inoculum doses and against a broader spectrum of fungal pathogens than strain Q8r1-96 or the phenazine gene donor strain. Despite having been known for over a century, the reactions involved in phenazine synthesis have remained obscure. ARS scientists and two groups of collaborators identified key genes, intermediates and novel steps in the biosynthetic pathway, opening new opportunities to improve the performance of phenazine-producing biocontrol agents.  Genes unique to D-genotype strains as a first step to defining molecular determinants of their unique affinity for wheat roots and superior rhizosphere competence were also identified. 

Impact:  These studies show that entire pathways for the synthesis of antifungal metabolites can be combined to generate superior biological control agents. Our strains are the basis of the longest-running risk assessment analysis of the effects of recombinant biocontrol bacteria in the United States. These strains have had no impact on non-target indigenous microbial populations in the rhizosphere of wheat, which may help to alleviate concerns about the introduction of recombinant strains into the environment. The strains also are a valuable research tool to identify and compensate for local factors limiting to the performance of introduced biocontrol agents. Although the antibiotic properties and pharmacological potential of phenazine antibiotics have been recognized for over a century, our research is the first in over 30 years to provide new insight into key reactions involved in their synthesis. The results will be used to target phenazine production more specifically to particular plant pathogens. Some phenazines produced by animal and human pathogens are virulence factors, and others have anticancer activity; our results also therefore are broadly applicable in drug design and disease treatment. 

Additional Information:  Competitive grants supported research that contributed to this accomplishment: 

O. A. Vogel. Wheat Research Fund.

Documentation:

Mavrodi, D. V., Bonsall, R. F., Delaney, S. M., Soule, M. J., Phillips, G., Thomashow, L. S. 2001. Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1. Journal of Bacteriology. V. 183 p. 6454-6465.

Mavrodi, O. V., McSpadden Gardener, B. B., Mavrodi, D. V., Bonsall, R. F., Weller, D. M. Thomashow, L. S. 2001. Genetic diversity of phlD from 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas species. Phytopathology. V. 91. p 35-43.

McDonald, M., Mavrodi, D. V., Thomashow, L. S., Floss, H. G. 2001. Phenazine biosynthesis in Pseudomonas fluorescens:  branchpoint from the primary shikimate biosynthetic pathway and role of phenazine-1,6-dicarboxylic acid. Journal of the American Chemical Society. V. 123 p.9459-9460.

Blankenfeldt, W., Kuzin, A., Skarina, T., Korniyenko, Y., Tong, L., Bayer, P., Janning, P., Thomashow, L. S., Mavrodi, D. V. 2004. Structure and function of the phenazine biosynthetic protein PhzF from Psesudomonas fluorescens. Proceedings of the National Academy of Sciences of the USA. V. 101 p.16431-16436.

Huang, Z., Bonsall, R. F., Mavrodi, D. V., Weller, D. M., Thomashow, L. S. 2004. Transformation of Pseudomonas fluorescens with genes for biosynthesis of phenazine-1-carboxylic acid improves biocontrol of Rhizoctonia root rot and in situ antibiotic production. Federation of European Microbiology Societies Microbiology Ecology. V. 49 p. 243-251.

Molecular interactions between Pseudomonas fluorescens and wheat roots

Background:  Biological control offers a sustainable means of controlling fungal soilborne pathogens of small grain cereals, particularly in the face of emerging root diseases and in the absence of effective genetic resistance in the host. Biocontrol isolates of the rhizobacterium Pseudomonas fluorescens exhibit preferences for specific crop species, and differ in their abilities to persist in the rhizosphere. P. fluorescens genes that mediate rhizosphere competence have been isolated, but contributing host-derived genes and mechanisms are unknown. Furthermore, the action of P. fluorescens-derived antifungal metabolites such as 2,4-diacetylphloroglucinol (DAPG) on host molecular and cellular processes remain poorly characterized.

Accomplishment:  Twenty-eight Pacific Northwest cultivars of hexaploid wheat were evaluated for their ability to undergo and sustain root colonization by two P. fluorescens isolates that vary in rhizosphere competence. Five cultivars supported higher rhizosphere populations of the aggressive root colonizing isolate Q8r1-96 (BOX-PCR genotype D) as compared to the less aggressive isolate Q2-87 (BOX-PCR genotype B), whereas other cultivars supported either high or low population sizes of both isolates. The phenotypes of the two P. fluorescens strains are virtually identical, and differ only in their molecular fingerprint. The findings were used to select “model” cultivars for subsequent studies of gene expression and DAPG accumulation. A microarray-based approach was used to determine if defense gene homologs of the model plant Arabidopsis are induced or repressed in wheat roots during colonization by P. fluorescens. Genes encoding a regulator of jasmonate signaling and proteins involved in oxidative stress were induced in wheat roots upon bacterial colonization. Changes in expression of the genes were authenticated using real-time PCR.

Impact: The findings demonstrate that host factors contribute to the rhizosphere competence of introduced rhizobacteria, and lay the foundation for genetic and molecular approaches to dissecting the relevant pathways and genes in wheat. This research also provides the first insight into two root-localized defense pathways triggered by aggressive biocontrol isolates, and suggests other mechanisms by which disease suppressiveness by biocontrol bacteria are achieved in wheat. The pathways will be a starting point for enhancing the efficacy of rhizobacteria in an important crop plant.

Documentation:

Okubara, P.A., Kornoely, J.P., Landa, B.B. 2004. Rhizosphere colonization of hexaploid wheat by Pseudomonas fluorescens strains Q8r1-96 and Q2-87 is cultivar-variable and associated with changes in gross root morphology. Biological Control. V. 30 p. 392-403.

Systematics of Biocontrol Fungi: Trichoderma and Hypocrea

Background:   Cacao is a tropical crop grown in almost all tropical regions. Its major economic effect in the United States is in the secondary products that are used in the preparation of chocolate and chocolate products. These include, but are not limited to, milk, wheat, nuts and fruit. Cacao is beset with two major diseases in tropical America and one major disease in West and Central Africa. These are witches’ broom disease and frosty pod rot in America and black pod caused by Phytophthora megakarya in Africa. Chemical controls for these diseases are of limited effectiveness and are only economically useful when the price of cocoa is high. The use of chemical controls removes the crop from the growing organic market while, at the same time, degrading the environment. Biological control alternatives are being sought that can either protect the crop (through induced resistance or a direct means) or can reduce the amount of inoculum.

Accomplishment:  This work has developed the ability to identify quickly unknown isolates of Trichoderma and to recognize undescribed species. A phylogeny of the genus Trichoderma has been developed using DNA sequencing and phenotype characterization. Partial monographs for groups of species of Trichoderma have been published. Isolates of Trichoderma have been received for identification from plant pathologists all around the, primarily, tropical world. Five have been shown to be effective in crop management, viz. T. asperellum against Phytophthora megakarya in cacao on Cameroon, the new species T. ovalisporum protecting against frosty pod rot in Costa Rica and two new species effective as protectants against frosty pod rot in Ecuador.

Impact:  The newly described species T. stromaticum is the active agent in the patented product TRICHOVAB, effective in reducing inoculum potential of the Witches’ Broom pathogen in Bahia, Brazil.  Prior to the development of a multigene phylogeny for Trichoderma species names were not accurately applied with the effect that communication about Trichoderma species could not be effective.  For example, our recent work on systematics of the T. koningii species complex species demonstrated that many reports in the literature of T. koningii used in biological control are based on misidentifications. The biological control species T. ovalisporum and one of the new species from Ecuador were initially identified as T. koningii.

Additional Information: Outside collaborations were significant and essential. Outside collaborators included USDA-ACSL lab (now SPCL lab), MasterFoods, USA; INIAP in Ecuador, IRAD in Cameroon, CABI-Bioscience in UK, IPARC in UK and CATIE in Costa Rica. Outside collaborators provided isolates and undertook field trials with identified strains.

Documentation:

Holmes, K., Schroers, H.-J., Thomas, S. E., Evans, H. C., Samuels, G.J.. 2004. Taxonomy and biocontrol potential of a new species of Trichoderma from the Amazon basin of South America. Mycological Progress 3: 199-210.

Chaverri, P., Samuels G. J. 2003.  Hypocrea/Trichoderma (Ascomycota, Hypocreales, Hypocreaceae):  Species with green ascospores. Studies in Mycology 48:1-116.

Chaverri, P., Castlebury, L. A., Samuels, G. J., Geiser, D. M. 2002. Multilocus phylogenetic structure of Trichoderma harzianum/Hypocrea lixii complex. Molecular Phylogenetics and Evolution 27:  302—313.

Samuels, G. J., Dodd, S. L., Gams, W., Castlebury, L. A., Petrini, O.  2002. Trichoderma species associated with the green mold epidemic of commercially grown Agaricus bisporus. Mycologia 94: 146-170.

Samuels, G. J., Pardo-Schultheiss, R., Hebbar, P., Lumsden, R. D., Bastos, C. N., Costa, J. C., Bezerra, J. L. 2000. Trichoderma stromaticum, sp. nov., a parasite of the cacao witches broom pathogen. Mycological Research 104: 760-764

Discovery Area 6:  Biocontrol of Insect Vectors of Pierce’s Disease

Novel viral pathogen of sharpshooter

Background:  Naturally occurring pathogens greatly reduce insect populations but may go unnoticed.  Discovery and characterization of new insect pathogens will aid management programs; combining these discoveries with the elucidation of insect genes that regulate critical developmental stages may lead to more efficient plant/insect pest management strategies.

Accomplishment:  New sharpshooter viruses have been discovered and are being characterized.  A provisional patent application has been filed.  Approximately 20,000 EST have been produced and published from the GWSS the vector of Xylella caused diseases.  An additional 20 full length genes and proteins have been released into the NCBI public database

Impact:  The new discovery of insect viruses specific to sharpshooters has opened a new area of research and focused efforts to integrate these biological control agents into existing IPM programs.  Entomopathogenic viruses are likely to reduce sharpshooter populations and limit spread of the disease.

Additional Information:  Development of genetic markers from grapes for marker-assisted selection in has been a collaborative effort with the Florida A&M University.  ARS provided the expertise to conduct EST sequencing and marker identification, and the Florida A&M University’s responsibility is to screen and characterize the markers within his grape breeding program.

Documentation:

Hunter, W.B., Lapointe, S.L., Sinisterra, X.H., Achor, D.S., Funk, C.J.  2003.  Iridovirus in Diaprepes Root Weevils (Coleoptera: Curculionidae: Diaprepes abbreviatus).  Journal of Insect Science 3:9.

Hunter,  W.B., P.M. Dang, M.G. Bausher, J. X. Chaparro, W. McKendree, R.G. Shatters, Jr., C.L. McKenzie and X.H. Sinisterra. 2003.  Aphid Biology: Expressed Genes from Alate Toxoptera citricida (Kirkaldy), Brown Citrus Aphid (Hemiptera: Aphididae). 7pp. Journal of Insect Science 3:23. 

Shatters, RG, Jr., Bausher, MG,  Hunter, WB, Chaparro, JX, Dang PM, Niedz, RP, Mayer, RT, McCollum, TG, and XH. Sinisterra. 2003.  Putative protease inhibitor gene discovery and transcript profiling during fruit development and leaf damage in grapefruit (Citrus paradise Macf.).  GENE 326: 77-86.

Hunter, W.B., C.P. Patte, X.H. Sinisterra, D.S. Achor, C.J. Funk and J.E. Polston. 2001. Discovering new insect viruses: Whitefly iridovirus (Homoptera: Aleyrodidae: Bemisia tabaci). Journal of Invertebrate Pathology 78: 220-225).

Funk, C.J.,  W.B. Hunter and  D. S. Achor. 2001. Replication of Insect Iridescent Virus 6 in a Whitefly Cell Line. Journal  of Invertebrate Pathology, 77(2): 144‑146.

 Cultural Control Component

Background:  Cultural methods for managing plant diseases are often inadequately exploited.  Improved cultural practices offer a general strategy for disease management with minimal environmental impact.  Knowledge of how tillage practices, crop rotations, water management, and other practices affect pathogen survival, disease initiation, and disease spread is needed to obtain the maximum benefits.

Discovery Area 1:  Natural Products and Soil Amendments Reduce Pathogen and/or Disease Development

Natural products and soil amendments are inadequately utilized sources of disease reduction technologies.  ARS scientists discovered a variety of products and amendments with application in reducing plant disease in the following areas.

Undiagnosed disorders in pecan orchards have become increasingly frequent and severe in recent years.  These maladies have also evolved into a replant disease issue affecting orchard establishment efforts.  The cause of the disorder has over the decades been attributed to any one of a host of biotic and abiotic agents.

Nematodes cause nearly ten billion dollars in crop losses per year in the United States and nearly $100 billion globally. For some crops, such as soybeans, nematodes are the most important pest or pathogen. However, many of the conventional nematicides that were used to control plant-parasitic nematodes have been shown to contribute to groundwater contamination and to be hazardous to the health of humans and animals, and have therefore been banned or restricted in use. It is particularly notable that the restrictions on methyl bromide application will remove the most widely used nematicide from agricultural use.  Consequently, new, effective and environmentally safe tools for managing nematodes are urgently needed.  ARS scientists are developing nematode management systems based on strategies that include identifying natural products that adversely affect nematodes and applying soil amendments to reduce nematode crop losses.

Greenhouse floricultural crops represent one of the largest commodities of American agriculture.  Improvements in greenhouse crop culture are required to enable American producers to remain competitive in world markets.  A joint research project, between the ARS and the University of Toledo (UT), is focused on hydroponic and soilless crop culture addressing the most important greenhouse stress management, disease, and insect problems as determined through interaction with the commercial greenhouse production industry.  Proper management of crops, diseases, and insect problems will enhance profitability and competitiveness of American growers. 

Pecan disorders traced to nickel deficiency

Accomplishment:  It was discovered that the cause of a growth malady (mouse-ear) and a replant disease, or disorder, that has become increasingly common in commercial pecan orchards, is a nickel (Ni) deficiency.  It was also found that Ni deficiency is largely due to excessive application of standard fertilizer nutrient elements to orchards (e.g., Ca, Mg, Fe, Mn, Cu, and Zn).  Thus, it was found that there has been excessive long-term fertilization of several mineral elements, especially that of zinc.  A Ni management strategy was developed that enables farmers to protect against economic losses associated with Ni deficiency^. 

Impact:  Nickel deficiency was discovered for the first time to actually exist in real-world agriculture, thus raising the possibility that many other agricultural crops suffering from replant problems or undiagnosed maladies may in fact be associated with a Ni deficiency being induced by fertility practices or root pathogens.  The work has affirmed that Ni is indeed an essential plant nutrient, has drawn attention to the fact that Ni nutrition of plants merits monitoring, has promoted marketing of Ni fertilizers, has promoted state regulatory bodies to authorize Ni usage in U.S. agriculture, and has produced a Provisional Pending Patent on Ni usage. 

Additional Information:  Outside funding from The Georgia Agricultural Commodity Commission for Pecans provided monetary support for this project research.  

Documentation:

Wood, B. W., Reilly, C. C., Nyczepir, A. P.  2004.  Mouse-ear of pecan: A nickel deficiency.  HortScience 39(6):1238-1242.

Wood, B.W., Reilly, C.C., Nyczepir, A.P. 2004. Mouse-ear of pecan: I. symptomology and occurrence. HortScience. 39(1):87-94.

Wood, B.W., Reilly, C.C., Nyczepir, A.P. 2003. Cu and Ni deficiency symptoms and mouse-ear in pecan.  HortScience. 38(5):726.

Alkaline-stabilized biosolid discovered to be effective against SCN and RKN as a soil amendment

Accomplishment:  The incorporation of organic amendments into agricultural soils offers potential benefits for both nematode control and improvement of soil fertility. Therefore, ARS scientists and collaborators evaluated the effect of an alkaline-stabilized biosolid soil amendment on SCN and RKN and on soil chemical properties, and investigated factors that may influence the efficacy of the amendment for suppressing plant-parasitic nematodes. The amendment was suppressive against both nematode species, and nematode suppression was positively correlated with pH, calcium, sulfur and ammonia concentrations in the soil solution.  Replacement studies demonstrated that an increase in soil pH was the soil chemical characteristic most closely related to nematode suppression, with ammonia production being secondary.  Studies also demonstrated that alkaline stabilization of biosolids was necessary to achieve nematode suppression, that microbes associated with the amendment appeared not to be responsible for the nematode suppressive activity, that alkaline-stabilized biosolids from different processing facilities all provided nematode suppression, and that the ability of this amendment to suppress RKN on tomato was influenced by soil type.  Additionally, several temperate and tropical cover crops are being evaluated for nematode suppression in vegetable, grape and soybean production systems. 

Impact:  The research is important because it provided an understanding of the mode of nematode suppression of an alkaline-stabilized biosolid amendment, and of factors that influence the efficacy of the amendment.  The ability of this amendment to suppress nematodes in a growers’ field will depend on soil type.  The work also indicated that alkaline-stabilized biosolids from several geographic locations were effective in suppressing plant-parasitic nematodes.  This will allow for the widespread use and marketing of this product as a consistent and reliable nematode management option.  Research evaluating cover crops for plant-parasitic nematode suppression have identified the toxicity of specific plant-derived compounds against nematodes potentially leading to the more reliable use of these plants as nematode management tools. 

Additional information:  ARS scientists collaborated with researchers at the following locations: 1) N-Viro International, Toledo, Ohio; University of Florida, Homestead, Florida; University of Michigan, East Lansing, Michigan; University of Iowa, Ames, Iowa; North Carolina State University, Raleigh, North Carolina; Ohio State University, Columbus, Ohio; and University of Manitoba, Winnipeg, MB, for alkaline-stabilized biosolids research; and 2) Pennsylvania State University, Bigglerville, Pennsylvania; University of Maryland, College Park, Maryland; and University of Florida, Homestead, Florida for the identification and evaluation of the chemical components of cover crops toxic to nematodes.

Documentation:

Zasada, I.A. and M. Tenuta.  2004.  Chemical-mediated toxicity of N-Viro Soil to Heterodera glycines and Meloidogyne incognita.  Journal of Nematology 36:297-302,

Chitwood, D.J.  2002.  Phytochemical based strategies for nematode control.  Annual Review of Phytopathology 40:221‑249.

Commercial greenhouse protocols studied

Accomplishment:  ARS scientists developed an understanding of the commercial greenhouse industry through personal visitations to build stakeholder rapport and to identify the priority, researchable problems facing growers.  Initial research studies have included evaluation of water-holding polymers in growing media, critical evaluation of specific nutrients (N, P, Fe and B), the uptake and fate of Si, and the effect of fertilizer anion species on growth, development, and disease expression.  Initial findings have been reported back to the grower community through informal grower-based meetings and poster presentations at national scientific meetings.  Major equipment purchases and installation has outfitted this startup site with the facilities necessary to conduct plant growth research to address the needs identified in the commercial greenhouse industry.  Additional support staff has been hired and outside researchers, both at the University of Toledo (UT) and other universities, have been identified to support specific aspects of the research through Specific Cooperative Agreements.  A regional Liaison Committee consisting of greenhouse producers and farm market operators, Cooperative Extension personnel, and allied stakeholders has been formed to provide assistance in understanding priority research needs, give knowledgeable feedback, provide a mechanism for technology transfer, and give credibility to the research mission.

Impact:  The infrastructure for this new project and rapport with the immediate stakeholders has been established, significant research partnerships have been formalized through Specific Cooperative Agreements and research productivity is occurring with initial technology transfer activities established through both scientific channels and directly to stakeholders, all of which will contribute to the goal of improving greenhouse crop production.  These improvements in production technology are being transferred directly to the stakeholders and will lead to enhanced profitability and competitiveness.

Additional information:  Specific Cooperative Agreements (SCAs) have been established during this past year with non-ARS scientists located at the University of Toledo (UT), North Carolina State University (NCSU), University of Florida (UF), and Ohio State University (OSU).  The SCA with UT is focusing on molecular mechanisms of plant response to abiotic and biotic stress that will lead to methods of rapid, non-destructive stress determination in plants in order to address the problem during the production cycle to yield high value crops.  The SCA with NCSU is directed to look at nutritional and pH relationships during production of geranium, the most important bedding plant species in American floriculture.  The SCA at UF is collaborating efforts to evaluate the uptake and potential role of silicon in bedding plants for crop improvement and disease resistance.  The SCA at OSU is focused on determining the role of the environment in nutrient uptake and partitioning in petunia.  

This greenhouse-based project was initially funded in FY 2001, but since it was directed to be established at a previously non-ARS location, the project had to be completely established with no staff, laboratory, equipment, or even a greenhouse.  The two ARS scientists were recruited and hired in FY 2002-2003 and support hires were made in FY 2003-2004. 

Documentation:

Bowers, J.H. and Locke, J.C.  2004.  Effect of formulated plant extracts and oils on population density of Phytophthora nicotiannae in soil and control of Phytophthora blight in the greenhouse.  Plant Dis. 88:11-16.

Frantz, J.M., Pitchay, D.S., Locke, J.C., Horst, L.E. and Krause, C.R.  2005.  Silicon is deposited in leaves of New Guinea Impatiens.  Online.  Plant Health Progress doi:10.1094/PHP-2005-0217-01-RS.

Locke, J.C., Pitchay, D.S. and Frantz, J.M.  2004.  Effect of nitrogen, potassium and silicon nutrition on disease susceptibility of various ornamental crop species.  Phytopathology 94:S62.  (Abstr.)

Frantz, J.M., Pitchay, D.S., Locke, J.C., Horst, L.E. and Krause, C.R.  2004.  Evaluating silica uptake in bedding plants.  HortScience 39(4):776.  (Abstr.)

Pitchay, D.S., Frantz, J.M., Locke, J.C. and Krause, C.R.  2004.  Managing nitrogen, potassium, and boron in bedding plants: Is more better?  HortScience 39(4):856.  (Abstr.)

Discovery Area 2: Chemical Treatments Impact Pathogen and/or Disease Development

Chemical alternatives to methyl bromide and new uses for proven chemical control compounds are highlighted in this area of ARS research.  Several projects focus on determining improved management strategies using chemicals for important diseases of small fruits, nuts and deciduous tree crops.  Phosphonate treatment for reducing Perennial Phytophthora Canker on almonds is reported as is the use of chloropicrin to replace methyl bromide in combating Prunus Replant Disease (PRD) on almond and peach.   

ARS research to determine effective fungicides for management of Sclerotinia in sunflower and dry edible bean was conducted as part of The National Sclerotinia Initiative.  The Initiative is a coordinated research effort aimed at reducing the economic threat of Sclerotinia in sunflower, soybean, canola, dry edible beans, chickpeas, lentils, and dry peas.  More details regarding this consortium are presented in NP303 component IV report.

Pecan scab disease and shuck decline are key economically important diseases affecting the U.S. pecan industry.  Effective and economical control has been a major challenge for the southeastern sector of the industry.  This is because of the difficulty of preventing nut yield losses, damage to foliage, impact on alternate bearing, and the economic loss associated with several cover sprays.  Additionally, yield and tree loss to mistletoe is a severe problem in certain locations.

Due to increasingly stricter water-use and run-off regulations, greenhouse and nursery operations capture water run-off that inadvertently contains pathogens. Irrigation water pumped from the catchment ponds redistributes pathogens onto the crops.  Few disinfestants are economically suitable for killing pathogens when pumping a high gallonage of water, as is typical of greenhouse and nursery production.  Chlorine gas, the most prevalently used disinfestant, may be restricted by regulations in the future. Chlorine dioxide is a disinfestant that is commercially used by many industries including treatment of municipal drinking water but had not been evaluated for use in ornamental plant production systems.  In addition to being effective, a disinfestant must pose minimal potential to cause damage on the hundreds of plant genera grown by ornamental plant producers.

Perennial Phytophthora canker (PPC) reduced using phosphonate

Accomplishment:  ARS scientists determined the efficacy and persistence of phosphonate treatments to prevent PPC of almond in numerous replicated orchard trials.  The research determined optimized methods and timing for phosphonate treatments in the almond industry.

Impact:  Perennial Phytophthora canker has caused severe losses in many almond orchards of the San Joaquin Valley, and the identification and optimization of effective phosphonate treatments has provided valuable control measures to the almond industry.

Additional information:  The ARS scientist collaborated in team research funded by USDA CSREES that targeted evaluations of methyl bromide alternatives for strawberry and deciduous fruit and nut crop industries.

Documentation:

Browne, G.T., Viveros, M.A.  2005.  Effects of phosphonate and mefenoxam treatments on development of perennial cankers caused by two Phytophthora spp. on almond.  Plant Disease 89:241-249.

Browne, G.T., Wilcox, W.F., Latore.  2005.  Phytophthora crown and root rot.  In: Compendium of Grape Diseases. The American Phytopathological Society, St. Paul, Minnesota.

Fumigant alternatives to methyl bromide developed to combat Prunus Replant Disease (PRD)

Accomplishment:  Prior to determining the PRD causative agent(s) ARS scientists developed an integrated management strategy for PRD on almond and peach that incorporates the use of several fumigant alternatives to methyl bromide that are applied to individual tree planting site locations.  The most effective was chloropicrin, which prevented PRD at application rates equivalent to standard methyl bromide applications.  The efficacy of the alternative chloropicrin treatments has significant implications for the fruit and nut industries.   In addition, ARS scientists showed that short-term pre-plant fallowing and cover crop rotations are also effective in the prevention of PRD.  Finally, they have provided evidence that there is cross specificity between peach and grape PRD which will have significant implications both the Almond and Grape industries. 

Impact:  Prunus Replant Disease is a significant limiting factor to Almond Production in California which is more than a billion dollar industry in the state.  ARS scientists developed several cost effective control strategies for this disorder that  have been accepted by the industry.  In addition these new control measures no longer require the use of methyl bromide which was the industry standard as recent as a 3-4 years ago.  In addition, several control strategies have been developed which are independent of chemical fumigation.