BIOASSAYS - CRITICAL TO BIOCONTROL OF PLANT DISEASE I

Harvey W. Spurr, Jr'

Abstract: Bioassays are frequently used by plant pathologists for studies related to disease resistance. fungicides, nematicides, and biocontrol agents. Their use was perfected with the development of chemical control agents. Bioassays were standardized as a result of cooperation among individuals. committees, industry and professional societies. Bioassays for biocontrol of plant disease have not been extensively developed. Considerable variation and diversity often exists with these bioassays because of the difficulty of standardizing two microorganisms and a plant host along with environmental conditions. However, the progress ond experience recorded to date arc evidence that bioassays can be used successfully to discover, develop and monitor biocontrol agents and biocontrol systems. More research on bioassays for biocontrol is needed and will increase the development of biocontrol technology for plant disease.

Key Words: Bioassay. biocontrol agents, IJlant diseases. disease resistance. fungicides. nematicides.

J. Agric. Entomol. 2(1): Ll7-122 (January 1985)

In plant pathology. bioassays are used to discover and develop disease resistance, fungicides, nematicides. and biocontrol agents. Recently. the interest in biological control of plant disease has increased considerably. Although the number of practical "in use" or "commercially available" plant disease biocontrol systems are few. prospects for a rapid increase are good.

Bioassays for plant disease biocontrol agents are often tests or evaluation systems designed to efficiently screen microbial isolates for control of plant pathogens, usually fungi or bacteria, in a regulated environment. These bioassays may exclude the host (in vitro) or include it (in vivo). As with bioassays used to detect fungicidal activity in chemicals, results are extrapolated to field situations where it is expected that many or most of the potential candidates selected from bioassays will fail. The efficiency and low cost of laboratory bioassays make then essential. Thus, biological control of plant disease will not progress without flrst developing efficient and reliable laboratory bioassays. Some of the problems and limitations associated with the development of bioassays ..vill be discussed.

Uses. Plant disease biocontrol agents are usually discovered as a result of applying an empirical approach. i.e., screening. Bioassays are designed for screening or discovering biocontrol agents. Primary bioassays are usually qualitative in lJitro tests between potential microbial agents and plant pathogens. A spore germination or slide-germination test on microscope slides or in petri dishes is a very efficient bioassay capable of aiding the discovery of biocontrol agents. Secondary in uiuo bioassays with host and pathogen in a regulated environment will strengthen the likelihood that potential biocontrol agents have been identified.

1 Cooperntive investigations of Agriculturnl Re8eorch Ser.ice, U. S. Del"ll'tment of Ag.riculture. lind North Cnrolina State Unlvenit)·. Depllrtment of Plant 1'1Ilholol:}', Hnleigh. Pllpur No. 20[;7 uf the Journlll SerieH of the North Cllrolinll Agricultural I{osellrch SCl"Yico. Use of Lrade nurnUH does not imply it>! cndol1lumenl hy the USDA or Lhu North Carolinll A!:riculturlll Hesellreh Sel"Yice of thll Ilroduch Illlmed Or 8irnih.r ones not mentioned. rteceived for publiClltion 22 Mil)' 198~: IIceepled 1 Z'O"ember 19801.

2 nCHellrch I'lont I'llthologiu. Soulhum Region. ARS. USDA. Ouord Tohllce<,t Ruellrch Llloorulory. Ouord. KC 2756" 11180, Profeuor o( Plllnt PlIthology. Korth CIIJ'Olill1l State Ulliverllity, Rllleigh 27695·7616.

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Bioassays are also used to help select "more active" strains or caces of known biocontrol agents. The bioassay then becomes a tool for quantifying biological activity of 8 biocontrol agent, thereby characterizing its control potential Dosage­response curves can be generated from bio8ssays and used to compare the efficacy of biocontrol agents. Bioasssys are used also for monitoring the production, formulation, application and persistence of biocontrol agents.

Design. Bioassays Bre designed for specific purposes related to characteristics of a disease, method of application of the control agent, and its mode of action, e.g., designs differ if a disease is caused by a soilborne, seedborne or foliar pathogen. Also, the design to detect hyperparasites differs from designs for discovering competition, hypovirulence or induced resistance. Bioassay designs also must be reliable, capable of yielding results for statistical analysis, and simple enough to be performed easily in the laboratory.

Bioassays are designed for plant pathologists who visualize the employment and management of biological agents for plant disease control as an important strategy to add flexibility to disease management. These researchers design bioassays to control one specific pathogen or disease although their concept may have broader applications if proven successful. The concept to develop one bioagent to control one pathogen is usually retained in succeeding research. A major problem with this approach is that biological control in situ or in the natural habitat usually involves several interactions between nonpathogenic and pathogenic microbes and environmental factors. Also, different hypotheses govern studies of antagonism compared to induced resistance. Therefore, bioassay designs for plant disease biocontrol relate distinctly to the philosophy, expectations and interpretations of the designers.

Variations. Variability is the nature of biological systems. This characteristic, which is a key factor to the survival of microbes, is a major source of difficulty to biologists who try to obtain reliable measurements. For a bioassay to be considered reliable there must be rigid control and standardization of the materials and procedures employed. Bioassays employ biological organisms and physical­mechanical systems, which are the main sources of random error. For example, in the slide-germination test, the production of potential bioagents, pathogen spores, equipment and test procedures must be standardized. If a bioassay includes disease initiation, the host plant production must be standardized. Also, the criteria used for replication, evaluation, statistical analysis and interpretation of results must be standardized. Difficulties in decreasing variations in results from bioassays is a constant frustration to researchers. Since this is 8 part of natural systems variation it is handled by replication and statistical analysis. This is especially true in bioassays for biocontrol of plant disease because they often include two different microbes and a host plant. Since a main factor in controlling variability is standardization, 8 discussion of selected examples of standardization follows.

Diversity. The fact that bioassays are designed and used by individuals is indicative of the diversity that prevails. [n most instances when a plant pathologist considers using a bioassay designed by another plant pathologist, he tries it, then redesigns or modifies it to suit his research orientation. Diversity also results from types of diseases, host plants, cultural conditions and production requirements. Thus, the pathologist concerned with control of powdery mildew on apple foliage requires bioassays different from those for Pythium damping-off of peas. Add to

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this diversity the variation described above and it can be asked whether standardization oC bioassays, if possible, is a realistic goal?

Standardization. The Question of standardization Cor the slide germination method of evaluating protectant fungicides was reviewed by The American Phytopathological Society, Committee on Standardization of Fungicidal Tests (1943). This required 30 years of development by plant pathologists, many of whom had published individually on the needs and methods for standardizing this method. This committee considered biological and mechanical errors associated with this bioassay. They made detailed recommendations that included standardized treatment of glassware, the pathogen spores (culture medium, age. etc.), the fungicide, germination criteria and data comparisons (number of replications, dosage-response curves, error terms). This information was published as a guide Cor plant pathologists and perhaps as a signal to be cautious when making changes or modifications in this bioassay if you expect your data to be accepted or respected.

Another response to the need for standardization of chemical bioassays was the 1978 publication, Methods for Evaluating Plant Fungicides, Nematicides, and Bactericides (The Amer. Phytopathological Sac. and Soc. Nematologists 1978). This hook was prepared jointly by members of The American Phytopathological Society and the Society of Nematologists with assistance from the E 35 Pesticides Committee and the E 35.16 Committee on Nematode Control the American Society Cor Testing and Materials. It provides general suggestions and guidelines for performing bioassays, as well as numerous examples of laboratory, greenhouse and field test procedures involving a large and diverse group of pathogens and hosts.

When the research and development of the insect pathogen, Bacillus thuringiensis (Bt) progressed to the point where Bt was being produced internationally in large Quantities both by research laboratory and by industry for commercial formulation and application, there was a tremendous need for 8 stardardized bioassay. Initially, a spore count was considered the best method for standardization of the Connulations. When it was realized that an endotoxin that Cormed at sporulation was insecticidal, Dulmage et al. (1971) proposed a standardized bioassay for formulations of Bt based on an international unit (IV). The bioassay required feeding formulations of Bt to lwvac of a selected strain oC the cabbage looper and comparing activity to 8 standard preparation of Bt. A detailed procedure was developed which included instructions for standardizing insects, test materials, replications, comparisons and calculation of IV's. Bergerjon and Dulmage (1977) expanded the discussion of Bt standardization needed by producers and users of commercial products, and international standardization focusing on comparison of products made in different countries by different processes and with different isolates of Bt

The considerable experience of entomologists and their associates with the standardization of microbial bioassays for biological control of insects is reviewed by Burges and Thomson (1971). They discuss types of bioassays, measures of potency, including concentration and viability, and procedures for standardization. Reading this review will benefit plant pathologists interested in biocontrol.

It may be concluded that plant pathologists as individuals must be concerned about the standardization of bioassays. When problems and concerns become large, committees of volunteers representing scientific societies and industries may

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agree to construct standards. It is interesting that the enforcement of these standard bioassays results from voluntary actions on the part of individuals or groups motivated by the need for a common language.

Bioassays for biocontrol of plant disease. In this section some specific problems and examples will be considered relative to the discovery and development of biocontrol of plant disease. Cook and Baker (1983) broadly defined biological control of plant pathogens 88 ... "any control achieved through a living system" (p. 83). They excluded man's activities as in the use of quarantines and the application of chemicals. This discussion of bioassays applies to the narrower definition, i.e., biocontrol research directed towards the discovery and development of specific microbes to control plant pathogens via specific interactions. Primary mechanisms include hyperparasitism, toxin formation, competition, and induced resistance. These approaches require a bioassay(s). This bioassay is more important than has been recognized in many instances. If you are seeking to develop a biocontrol system for a selected disease using microbial management as a method, how do you discover and develop the microbes and system? Aside from accidential discoveries, reliance is placed on an empirical approach: bioassays for screening. If it is agreed that considerable reliance is placed on bioassays, then it is necessary to question whether sufficient thought and effort are devoted to the development and use of bioassays by plant pathologists. I contend that most plant pathologists move too rapidly from initial discoveries in laboratory bioassays to more complicated environmental situations such as field tclsts. These tests in complex environments have eliminated most potential biocontrol agents (Spurr 1981a).

Our research on biocontrol of foliar disease and other biocontrol projects has shown the critical need for good bioassays. In a search for antagonists to foliar pathogens we used a slide-germination bioassay. Bacteria from standardized cultures were mixed with fungal spores in water, incubated on glass slides and germination was evaluated. Inhibition of germ tube formation, length of germ tube, number of branches and appressorium formation were criteria for evaluating antagonism. Thus, qualitative changes in germination (inhibition) at one dosage of bacteria were used as criteria for selecting antagonists. Fravel and Spurr (1977) selected five bacterial strains by this procedure and used them in a secondary laboratory bioassay that employed the pathogen and excised leaf tissue in a regulated environment (Spurr 1973). In this system, strains which were most inhibitory in vitro were also most inhibitory to the fungus on the leaf surface. The most active isolate was identified as Bacillus cereus var. mycoides, a common resident of leaves of many species in North Carolina. These observations stimulated additional screening of Bacillus species, including Bt, and led to the development of research with Bt for biocontrol of fungal leaf pathogens (SpUIT 1981b). We observed in this research that the number of candidates for control decreased as the complexity of the bioassay increased. This is a common phenomenon, which has not been adequately explained. In n greenhouse bioassay of bacterial isolates for control of cucumber anthracnose, Leben (1964) observed that one strain of the 230 tested inhibited disease, and none enhanced disease. This was a low frequency compared to the screening of bacterial isolates for control of tobacco Alternaria lenfspot where approximately 20% of the isolates tested demonstrated some activity (Fravel and Spurr 1977).

The in vitro bioassays of Fravel and Spurr (1977) showed that some bacterial strains significantly increased germ tube length and the number of germ tubes.

SPURR: Bio88s8Ys - Critical to Biocontrol 121

Although most researchers depend on inhibition as a criterion for selecting bioagents for biocontrol we considered the idea that stimulation of some pathogens could result in either an increase or decrease in disease. I have not seen a report wherein biological agents which stimulated pathogens in vitro were tested in vivo. Thus, the design of bioassays and the criteria for selection of candidates limit the scope for discovering and developing biocontrol agents.

Well-tested bioassays can usually be readily adapted for use in new projects aimed at biocontrol. Chen et aI. (1981) used an accepted agar incorporation bioassay for the detection of avirulent bacteriocin·producing strains of the soilborne bacterial wilt pathogen, Pseudomonas solanacearum. This bioassay resulted in the selection of several strains which were later used in field tests and provided partial control of bacterial wilt.

The importance of quantification and comparison of chemical agents via bioassays is well understood. Similar procedures can be used for comparing biocontrol agents and have been described and used with microbial insecticides (Burges and Thomson 1971). Spurr (l981b) emphasized that quantification is often ignored by plant pathologists, and microbial antagonists are selected on the basis of a Qualitative observation where only a single dosage is tested. In laboratory bioassays with several bacterial strains selected for their antagonism for foliar pathogens, it was observed that many had ED50 values of 109 colony forming units (CFU)/ml. Few had ED" values of 107 CFU/ml and none were observed to have ED,o values of 10' CFU/ml (Spurr, unpublished). In field tests only those bioagents with ED50 values of 107 CFU/ml or lower have resulted in 50% or more control of foliar disease (Spurr 1981b). A bioagent with an ED50 of 105 CFU/ml would have a better chance for success. A quantitative laboratory bioassay was used by Spurr (1977, 1979) to demonstrate that nonpathogenic conidia of Alternaria alternata applied to leaf surfaces could control leafspot caused by pathogenic A. aLtemata without interacting with other leaf surface microorganisms. This does not mean that leaf surface interactions between the protective fungus and the pathogen do not occur and thereby alter control efficacy. Thus, Quantitative bioassays can be used in many ways in biocontrol research.

Conclusions: Bioassays are much used by plant pathogists and have developed with the field of phytopathology. Their use was perfected in the development of chemical control agents. Bioassays were standardized as a result of cooperation among individuals, committees, industry and professional societies stimulated by a common goal. Bioassays for biocontrol of plant disease have not been developed or utilized to the extent possible thereby limiting the development of biocontrol for plant disease. The progress and experience to date are evidence that bioassays are critical to biocontrol of plant disease and can be used successfully to discover and develop biocontrol agents and biocontrol systems.

ACKNOWLEDGMENTS

I thank Eleanor T. Howard, Linda D. Daniel. and G. R. Knudsen for Lcchnical assistance.

122 J. Agrie. Entomol. Vol. 2, No. I (1985)

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