6

Answers to Charge Questions

On the basis of the reviews presented in Chapters 2-5, the committee concluded that the available studies of Fusarium oxysporum f.sp. cannabis, F. oxysporum f.sp. erythroxyli, and Crivellia papaveracea or Brachycladium papaveris are preliminary and exploratory. Thus, in answering the specific charge questions, the committee attempted to capture what conclusions could be drawn from the limited dataset and what might be expected on the basis of what is known about fungal pathogens and mycoherbicides in general.

EFFECTIVENESS

Are the drug crops (cannabis, coca, and opium poppy) known to be susceptible to the proposed mycoherbicides?

img The committee found documented evidence from greenhouse and small field studies that cannabis (Cannabis sativa), coca (Erythroxylum coca and E. novogranatense), and opium poppy (Papaver somniferum) are susceptible to F. oxysporum f.sp. cannabis, F. oxysporum f.sp. erythroxyli, and C. papaveracea/B. papa-veris, respecttively. However, the few studies available involved controlled applications to the target plants and often used artificial environmental conditions that favored infection. Thus, the committee judged that the information is insufficient to conclude that the proposed mycoherbicides would be efficacious for the control of illicitdrug crops under field conditions.

Have the mechanisms of action of the proposed mycoherbicides’ toxicity to illicit-drug crops been established?

img The types of diseases produced by the proposed mycoherbicide strains are wilt diseases in cannabis and coca and a blight of the aerial parts of opium poppy. Some members of the fungal species to which the proposed mycoherbicide strains belong can produce toxins



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6 Answers to Charge Questions On the basis of the reviews presented in Chapters 2-5, the committee con- cluded that the available studies of Fusarium oxysporum f.sp. cannabis, F. ox- ysporum f.sp. erythroxyli, and Crivellia papaveracea or Brachycladium pa- paveris are preliminary and exploratory. Thus, in answering the specific charge questions, the committee attempted to capture what conclusions could be drawn from the limited dataset and what might be expected on the basis of what is known about fungal pathogens and mycoherbicides in general. EFFECTIVENESS  Are the drug crops (cannabis, coca, and opium poppy) known to be susceptible to the proposed mycoherbicides? o The committee found documented evidence from greenhouse and small field studies that cannabis (Cannabis sativa), coca (Erythroxy- lum coca and E. novogranatense), and opium poppy (Papaver som- niferum) are susceptible to F. oxysporum f.sp. cannabis, F. ox- ysporum f.sp. erythroxyli, and C. papaveracea/B. papa-veris, re- specttively. However, the few studies available involved controlled applications to the target plants and often used artificial environ- mental conditions that favored infection. Thus, the committee judged that the information is insufficient to conclude that the pro- posed mycoherbicides would be efficacious for the control of illicit- drug crops under field conditions.  Have the mechanisms of action of the proposed mycoherbicides’ toxicity to illicit-drug crops been established? o The types of diseases produced by the proposed mycoherbicide strains are wilt diseases in cannabis and coca and a blight of the ae- rial parts of opium poppy. Some members of the fungal species to which the proposed mycoherbicide strains belong can produce tox- 121

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122 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops ins involved in disease development under laboratory conditions, but no reliable data are available on the proposed mycoherbicide strains. The molecular mechanisms underlying the host-pathogen interac- tions are unknown, and the nature of disease progression and fungal spread after disease onset, which are critical determinants of myco- herbicide efficacy, are not documented.  Are the proposed mycoherbicides host-plant-specific? o The proposed mycoherbicide strains can cause disease in their target plants, including those grown legally and those which occur natu- rally near the illicit crops. However, the few host-range studies con- ducted with nonrelated species were of little value because they only report that the mycoherbicide strains did not cause disease in some native plants and crop species and do not provide experimental de- tails (or in some cases even the names of the plants). Furthermore, none of the available studies used a standard, systematic process to select the most relevant plants to test in host-range studies. For ex- ample, of about 200 species of Erythroxylum native to South Amer- ica, only two were tested for susceptibility to the coca mycoherbi- cide. Thus, the data are insufficient to conclude that the proposed mycoherbicides would not pose a risk to other plants or crops.  What quantities of mycoherbicides would be needed to eradicate il- licit drug crops? o Workable mycoherbicide formulations, delivery methods, and appli- cation strategies need to be developed in partnership with an indus- trial producer before this question can be answered. The committee made estimates for a single application per hectare solely on the ba- sis of published laboratory production methods, which may or may not be realistic. Its estimates indicate that tens to hundreds of kilo- grams of dry formulations per hectare or hundreds to thousands of liters of liquid-spray formulation (containing billions to trillions of spores) per hectare would be required for a single application of the mycoherbicides.  How would the method of delivery affect the effectiveness of the mycoherbicides in eradicating the drug crops? o For all three mycoherbicides, on-ground application would be the most precise and uniform. However, it is not feasible, because the growers of illicit drug crops would be uncooperative and possibly hostile. Aerial application is probably the most practical approach, but it would need to be from altitudes greater than those normally used for agricultural applications.

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123 Answers to Charge Questions o F. oxysporum f.sp. cannabis and F. oxysporum f.sp. erythroxyli are soilborne, root-infecting pathogens; for greatest efficacy, they should be delivered to the soil. Aerial application of dry formula- tions, such as prills and pellets, could reduce the efficacy of such soilborne pathogens because the formulations would be subject to scattering by wind, which would lead to nonuniform, discontinuous placement of the inoculum over the target area and reduce the size of the plant-pathogen interface. o C. papaveracea/B. papaveris is a pathogen that attacks primarily the aerial parts of opium poppy; for greatest efficacy, it should be sprayed on the foliage of the plants. All tests of this pathogen have used a wet application. Aerial application of liquid formulations would be subject to wind-driven drift, which would lead to irregular deposition on the target area. The large amount of water needed for aerial delivery of the mycoherbicide could be an even more impor- tant limiting factor. Low-volume and ultra-low-volume applications have not been tried with the proposed mycoherbicides. FEASIBILITY OF LARGE-SCALE MANUFACTURE AND DELIVERY  What sort of facility would be required for the large-scale Indus- trial manufacture of the proposed mycoherbicides? o An entity with industrial-scale liquid or solid-state fermentation and formulation capabilities and capacity to produce sufficient tonnage of the finished products per year would be needed for large-scale manufacture of mycoherbicides. The experience gained in industrial production of registered mycoherbicides could be adapted to pro- duce the materials needed.  What sort of equipment and technology would be required for the delivery of the proposed mycoherbicides on a large scale? o Delivery of the mycoherbicides is expected to pose a challenge. The most likely scenario is aerial application of the proposed mycoherbi- cides from an aircraft capable of delivering a dry or liquid formula- tion. o On the basis of published data, the Fusarium mycoherbicides would be best delivered as dry formulations and the Crivellia/Brachy- cladium mycoherbicide as a spray. However, the latter appears im- practical because of the amount of water required for spraying and the possible inability to apply from low altitudes.  What is the overall technical feasibility of the large-scale industrial manufacture of the proposed mycoherbicides? o Technology for large-scale industrial production of commercial my-

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124 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops coherbicides against weeds exists. It probably could be adapted for production of the proposed mycoherbicides. However, there are no data on whether the proposed strains could be mass-produced on a large scale.  What consideration would need to be made for large-scale delivery of the proposed mycoherbicides? o In addition to the equipment and technology required for large-scale manufacture and application, consideration should be given to the potential impediments to implementing the use of the mycoherbi- cides. The impediments include the need for international approval and cooperation with the countries in which the mycoherbicides are to be used and the uncooperative and possibly hostile growers who might try to prevent or counteract the effects of mycoherbicide ap- plication.  What types of field trials of the proposed agents are needed? For example, is testing in tropical or arid environments required? o More epidemiological data on the proposed mycoherbicides from experiments with consistent protocols are needed before extensive field tests can be conducted. In general, the field-test conditions should mimic the climatic and environmental conditions under which the illicit-drug crops are grown, and the mycoherbicides should be applied in a manner that simulates the intended delivery method. o The published data shed some light on the environmental conditions that favor the mycoherbicides’ performance. This information is useful for guiding further research. There would have to be several on-site trials over a few years and in different areas where the drug crops are grown to identify field conditions that are conducive to the efficacious performance of the mycoherbicide and to establish the best timing for application. This phase of research should be done in cooperation with industry. o As part of any future research program, ecoclimatic data on major drug-producing regions should be gathered to select test sites and regions where the mycoherbicides could be tested and used success- fully. Climate-matching computer programs could help identify geographic regions where the climatic conditions would be condu- cive for the mycoherbicides to cause substantial damage on their target crops. PERSISTENCE IN THE ENVIRONMENT  How long are the mycoherbicides likely to persist in the environ- ment after application?

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125 Answers to Charge Questions o Persistence of the mycoherbicide fungi would depend on environ- mental conditions at the application site and whether the type of fungal inoculum (asexual or sexual spores, microsclerotia, pseudo- thecia, or mycelia) applied would be able to survive environmental stress. Simply determining the abiotic conditions that favor or re- duce persistence under controlled environments is insufficient be- cause persistence would also depend on interactions with other liv- ing organisms and on environmental conditions that might not be replicable in controlled settings.  Do the mycoherbicides have geographic or climatic boundaries? o F. oxysporum f.sp. cannabis, F. oxysporum f.sp. erythroxyli, and C. papaveracea/B. papaveris occur naturally in the native geographic ranges of their target crops. Particular strains probably have geo- graphic and climatic boundaries that would limit their survival and infectivity, but the strains that were tested were evaluated under par- ticular environmental conditions, so it is not possible to define those boundaries.  What combination of environmental conditions (such as tempera- ture, depth in soil, and pH) would favor persistence after application? o Moisture (from dew, high relative humidity, or rainfall) for several hours over several days with favorable temperatures would probably be required for the pathogens to become established on the target crops. o Information on moisture, temperature, and other requirements for pathogen survival are based on data on a few strains collected from very small areas relative to the entire geographic and climatic ranges of the target drug crops. The available data are not sufficient to con- clude that the strains studied would be effective in all areas where the illicit crops are grown. o Studies have not examined the survival of various types of fungal propagules (spores, mycelia, or other structures) either in general or derived from the particular strains studied for development as my- coherbicides.  What conditions would shorten the persistence of the proposed mycoherbicides in the environment? o In general, a variety of factors could shorten the persistence of fungi, such as moisture conditions (too much or too little moisture), ex- treme temperatures (too high or too low), ultraviolet radiation, soil depth and compaction, and the presence of antagonistic organisms, including other microorganisms, plants, and microfauna.

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126 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops  Could persistence of the mycoherbicides in the environment be controlled? o Shortening persistence: The persistence of mycoherbicides in the environment could be shortened by the application of chemicals, such as fungicides or soil fumigants. Such a control strategy would be effective for reducing mycoherbicide agents only in small areas and would be impractical or impossible for large areas. Controlling the mycoherbicides in this manner carries substantial risk of harm- ing the environment or other organisms in the treated area. o Prolonging persistence: The available data support the hypothesis that increased inoculum levels could be maintained for a few months after application but do not support the hypothesis that the mycoher- bicide strains could persist indefinitely at higher population densities than those of the indigenous strains of the same fungi. TOXICITY TO NONTARGET ORGANISMS  Would the proposed mycoherbicides harm licit crops or kill other soil fungi? o Licit crops of cannabis, coca, and poppies could be adversely af- fected if they were exposed to the proposed mycoherbicides. Such exposures could occur through accidental drift of aerially applied mycoherbicides; through transport by human, insect, or rodent carri- ers; through seed transmission; or through long-distance aerial transport of spores. The available data are insufficient to determine whether any of the proposed mycoherbicides would harm other types of licit crops, native plants, or soil microbiota.  Would the proposed mycoherbicides threaten biodiversity or pose other risks to the environment? o There are few data on risks posed by the application of the proposed mycoherbicides. To the extent that a large volume of fungal inocu- lum would be introduced by the application of the mycoherbicides, the biodiversity and ecology of the target regions could be affected. The risk of disease in related, nontarget species of Cannabis, Erythroxylum, and Papaver and to native plants caused by the my- coherbicide strains and the consequent indirect effects on microor- ganisms and fauna are unknown.  What is the nature of the health risks to animals and humans posed by the use of the proposed mycoherbicides? o There are no reliable data on the health risks posed by F. oxysporum f.sp. cannabis, F. oxysporum f.sp. erythroxyli, and C. papav- eracea/B. papaveris to animals or humans. Some strains of F. ox-

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127 Answers to Charge Questions ysporum can infect humans or produce low levels of mycotoxins under laboratory conditions, but the variation within this species complex and the relative rarity of human infections and known my- cotoxin-producing strains means that other strains in the species complex cannot serve as surrogates for the proposed mycoherbicide strains. o There are no data on the types or quantities of toxins or allergens produced, if any, by the proposed mycoherbicide fungi. Without such data, risks to human and animal health cannot be evaluated.  What would be the range of transmission of the proposed myco- herbicides and what factors would influence their spread? o There is potential for the spread of the mycoherbicides beyond the site of application by a variety of means, including dispersal by wind, rain, insects, animals, and humans and spread with seed or soil. Therefore, neither the range nor the speed of transmission of the proposed mycoherbicides can be predicted.  Once released, would the pathogens uncontrollable? o Control or containment of the mycoherbicide strains after they are released would be all but impossible. The fungal strains are living organisms that interact with and adapt to their environment. Their ability to survive, propagate, and disperse beyond the target area would depend on environmental factors that can be neither predicted nor controlled. The persistence of indigenous strains of the fungi across the native range of their hosts is consistent with the conclu- sion that introduced mycoherbicide strains are unlikely to be con- tained or eradicated once they are released. POTENTIAL FOR MUTATION AND RESULTING TOXICITY TO TARGET AND NONTARGET ORGANISMS  What would be the potential of the pathogens to mutate? o The potential of mycoherbicide fungi to mutate would be similar to that of fungi in general. The genomes of fungi could change by nu- cleotide substitution, the gain of genetic material either from closely related species (by introgression) or from distantly related fungi (by horizontal gene transfer), the duplication of genetic material, and the loss of genetic material. New genetic variation could become estab- lished in fungal populations by natural selection or by chance. Natu- ral selection results in adaptation to changing environments, includ- ing adaptation to new cultivars of the target crops or to new host species (nontarget crops). Adaptation might occur in fungi that re- produce sexually or asexually. Sexual reproduction allows new

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128 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops genotypes to be produced more quickly through recombination than does mutation alone, as would occur in asexually reproducing or- ganisms. o One place where the mycoherbicides would be expected to adapt would be during the fermentation necessary to produce large amounts of inoculum. In such a case, adaptation to the fermentation conditions might alter the virulence of the mycoherbicides. It seems likely that the alteration would be to lower virulence rather than to raise it. Thus, as in any industrial fermentation, quality control of both the product at the end of fermentation and the inoculum that is used to start the fermentation process would be essential. o F. oxysporum reproduces clonally; no sexual stage is reported. However, the distribution of mating type alleles and genotypic di- versity in soil populations of F. oxysporum are consistent with sex- ual recombination in field populations of this fungus. C. papav- eracea reproduces clonally and by sexual recombination. B. papaveris reproduces clonally and, although it also reproduces sexu- ally, is self-fertile; the relative proportions of inbreeding and out- breeding are unknown.  Are there environmental or other conditions that would drive mu- tations in the mycoherbicides? o Potential mutagenic changes in the proposed mycoherbicide strains should be the same as in any fungus. There is no reason to expect the proposed mycoherbicides to be more or less susceptible to muta- tion due to environmental or other conditions than indigenous strains.  Does the potential for mutation pose additional risks to nontarget organisms (including other plants, fungi, animals, and humans) or the envi- ronment? o It is not possible to predict what types of mutations that might ad- versely affect nontarget organisms would occur.  How might mutations affect the susceptibility of the target crops to the mycoherbicides? o Planting resistant crop plants selects for new fungal genotypes that can overcome the host plant’s resistance. Selection for crop varieties that are resistant to existing fungal genotypes follows, and the cycle repeats. Selection for resistant drug crop varieties would reduce the effectiveness of the mycoherbicides and would require continued development of new mycoherbicide strains that could attack the newly developed target crop genotypes.

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129 Answers to Charge Questions o Mutation of the proposed mycoherbicide strains could reduce, in- crease, or have no effect on the efficacy of the mycoherbicides on a target crop. It is not possible to predict what type of mutations might occur, how the pathogen or host plant might be affected, or whether the mutations would be favored by natural selection.  How might mutations affect the toxicity of mycoherbicides generally? o Given that F. oxysporum and C. papaveracea/B. papaveris are al- ready present where the illicit-drug crops are grown, there is no rea- son to expect that toxins, or mutations that affect toxins, found in the mycoherbicides would differ from those already present in indige- nous strains. RESEARCH AND DEVELOPMENT NEEDS  Could the effectiveness of the mycoherbicides in eradicating illicit- drug crops be improved through research and development? o Only a few strains of the proposed mycoherbicides considered in this report have been investigated. Before improvements can be con- sidered, more research is needed to obtain and identify the most ef- ficacious strains available (preferably from plants common to the target regions), the most susceptible stage in the life cycle of the tar- get crop, and the climatic and environmental conditions required for disease development. o Once those conditions are met, there are ways to improve the effec- tiveness of the mycoherbicides, such as the use of adjuvants and the development of formulation and delivery methods to facilitate and maximize infection.  What types of expertise would be most relevant for improving the effectiveness and safety of the proposed mycoherbicides? o At a minimum, a team with expertise in the growing practices and conditions under which the drug crops are grown; in plant ecology; in plant disease epidemiology; in plant pathology; in fungal genet- ics; in fermentation, formulation, and application technology; and in nontarget risk assessment is needed to develop the proposed strains as mycoherbicides, to assess their effectiveness and safety, and to gather data for Environmental Protection Agency (EPA) registra- tion. o Partnerships between industry and science are needed to guide tech- nical development and to assess the feasibility of producing the pro- posed mycoherbicides.

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130 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops  What types of research and technology would improve the produc- tion and delivery of the mycoherbicides? o Research is needed to determine whether the proposed mycoherbi- cide strains could be produced on a large scale. Research is also needed to guide product formulation, storability, and delivery. The technology to support production and delivery could be identified or developed as needed by an industrial producer.  What type of testing would be needed before mycoherbicides could be safely and effectively used to eradicate illicit-drug crops (for example, mode-of-action studies)? o A full complement of research documenting the efficacy, specificity, and mode of action of the proposed mycoherbicide products is needed. o For safety assessments, at a minimum, EPA’s toxicology, nontarget- organism, and environmental-fate data requirements for microbial pesticides should be met. o Testing in countries where the mycoherbicides would be used should be done, particularly to address local biodiversity and non- target-risk considerations.  What would be required under U.S. federal and state laws to test and approve a mycoherbicide of this type, and what guidelines of the Inter- national Organisation for Biological and Integrated Control of Noxious Animals and Plants1 would apply? o The committee did not feel comfortable in developing a checklist of testing requirements that must be met, especially with regard to state and international rules, which differ by state and country. The ex- perience from testing registered mycoherbicides suggests that the requirements become part of an evolving process as dictated by the experimental findings with regard to individual organism, host, and conditions. The committee therefore took a more general approach of reviewing and identifying the types of data required for registra- tion in the United States and the special considerations for domestic and international uses of the proposed mycoherbicides. 1 The International Organisation for Biological and Integrated Control of Noxious Animals and Plants is a professional society that promotes the use of biological control. While the organization and some of its members have been involved in the development of certain international standards for testing of pesticides and guidelines for transport and release of biological control agents, the IOBC does not have its own set of requirements. The committee interpreted this question to be a more general one about what interna- tional guidelines are relevant to mycoherbicides, and reviewed pertinent international treaties and guidance from the United Nations.

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131 Answers to Charge Questions o For a microbial pesticide to be registered by EPA for use in the United States, a variety of tests and reports are required, for exam- ple, on product analysis, pesticide residues, toxicology, effects on nontarget organisms, and environmental fate of the pesticide. Such testing is required for each strain of a mycoherbicide to be regis- tered. An environmental assessment or an environmental impact statement would probably be required for each mycoherbicide and perhaps for each strain. o At the international level, applicable guidelines and potential limita- tions for testing, approval, and application of mycoherbicides against illicit crops fall under the International Plant Protection Convention, the International Standards for Phytosanitary Measures, the Biological Weapons Convention of 1972, and legal requirements in the country where the mycoherbicides would be used. Collec- tively, the guidelines are intended to prevent the spread and intro- duction of plant pests by requiring their safe export, import, ship- ment, and release. Some of the requirements involve documentation for the transport of agents, conducting risk assessments, and imple- menting risk-management strategies.