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5 Crivellia papaveracea and Brachycladium papaveris as Candidate Biological Control Agents against Opium Poppy Fungal diseases are a normal occurrence where opium poppy (Papaver somniferum) is grown repeatedly. Among the fungal pathogens that can cause serious damage to poppy plants are species of Brachycladium, Crivellia, Fusarium, Peronospora, and Verticillium (Scott 1877; Harrison and Schmitt 1967; Dolgovskaya et al. 1996; Podlipaev et al. 1996; Reznik et al. 1996; Finetto 2008). Crivellia papaveracea and Brachycladium papaveris (formerly known as Pleospora papaveracea and Dendryphion penicillatum, respectively) have received the most attention as parasites that might substantially limit licit poppy cultivation (Milatović 1975a b) and as potential mycoherbicides against illicit opium poppy crop (Del Serrone and Annesi 1990; O’Neill et al. 2000; Bailey et al. 2000b, 2004a, 2004b; UNODC 2002). Both fungi are distributed worldwide wherever poppy is grown (Milatović 1975a; Munro 1978; Sivanesan and Holliday 1982). As noted in Chapter 1, a single fungus may have more than one name, and this can complicate interpretation of the scientific literature. Nowhere is that problem more evident than in the literature on C. papaveracea and B. papaveris. In this report, the publication by Inderbitzin et al. (2006) is used for the taxonomy of those fungi because it used DNA sequence data to identify strains, including many of the ones used in the studies cited in this chapter. Inderbitzin et al. (2006) describe how different authors have used the names in different ways. In the present report, where possible, we have translated the names used by the various authors into the currently accepted names, C. papaveracea and B. papaveris. Readers who consult the original literature may find Table 5-1 useful in attempting to equate the names used in that literature with the names used 101
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102 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops here. The attributes of C. papaveracea and B. papaveris are listed in Table 5-2 with the two isolates studied by O’Neill et al. (2000) as reference strains. When it is unclear which of the two species was studied, we refer to both in this manner: C. papaveracea/B. papaveris. C. papaveracea/B. papaveris was reported as a pathogen of poppy by Milatović (1975a,b) and Czyzewska and Zarzycka (1960). C. papaveracea and B. papaveris are seedborne pathogens that are widely distributed around the world wherever poppy is grown but are most commonly observed in Europe and Asia (Schmitt and Lipscomb 1975). Both can cause serious damage to poppy when conditions for disease are optimal (Krikorian and Ledbetter 1975; Schmitt and Lipscomb 1975). C. papaveracea/B. papaveris attacks plant roots and parts above the ground; disease symptoms include seedling damping-off, girdling of roots, and lesions on leaves, stems, and capsules (Meffert 1950; O’Neill et al. 2000). TABLE 5-1 Fungi Used in Various Papaver Mycoherbicide Studies Identification Given Identification by Fungal Strain (Reference) in Reference Inderbitzin et al. 2006 Strain 7359 (Meffert 1950) Dendryphion penicillatum Crivellia papaveracea Strain 3 (Meffert 1950) Helminthosporium papaveris Brachycladium papaveris Isolates used by Del Serrone Dendryphion state of Strains not studied by and Annesi 1990 P. papaveracea Inderbitzin et al. 2006. Inferred to be a heterothallic fungus. Strain Cf96 (Farr et al. 2000) Dendryphion penicillatum Crivellia papaveracea Strain Pf96 (Farr et al. 2000) Pleospora papaveracea Brachycladium papaveris Strain Cf96 (O’Neill et al. 2000) Dendryphion penicillatum Crivellia papaveracea Strain B96 (O’Neill et al. 2000) Pleospora papaveracea Brachycladium papaveris Strain Pf96 (Bailey et al. 2000a, Pleospora papaveracea Brachycladium papaveris 2000b) Strain Cf96 (Bailey et al. 2000b) Dendryphion penicillatum Crivellia papaveracea No strain number; inferred to Pleospora papaveracea Brachycladium be Pf96 (Bailey et al. 2004a) papaveris (if the isolate is indeed Pf96) Strain Pf96 (Bailey et al. 2004b) Pleospora papaveracea Brachycladium papaveris Strain C-6-3 (UNODC 2002) Pleospora papaveracea (Not studied by Inderbitzin et al. 2006)
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103 Candidate Biological Control Agents against Opium Poppy TABLE 5-2 Attributes of the Papaver Mycoherbicides Crivellia papaveracea and Brachycladium papaveris Crivellia Brachycladium Attribute papaveracea papaveris Reference Reference straina Cf96 Dendryphion Pf96 Pleospora O’Neill et al. 2000 penicillatum papaveracea None describedb Teleomorph (sexual form) Crivellia Inderbitzin et papaveracea al. 2006 Anamorph (asexual form) Brachycladium Brachycladium Inderbitzin et penicillatum papaveris al. 2006 Sexual reproduction Heterothallic Homothallic Inderbitzin et (requires mating (self-mating) al. 2006 partner) Macroconidiophoresc Produced Not produced Inderbitzin et al. 2006 Microsclerotiad Present Absent Inderbitzin et al. 2006; Meffert 1950 Chlamydosporese Not reported Infrequent; intercalary Farr et al. 2000; (between apex and base) Meffert 1950 Pseudotheciaf Present in field Present in field material; O’Neill et al. 2000 material produced by laboratory cultures older than 30 days Relative virulence Less virulent More virulent Bailey et al. 2000b; O’Neill et al. 2000 a Strains used by O’Neill et al. 2000 and studied by Inderbitzin et al. 2006. b The situation with B. papaveris is unusual because this fungus has a sexual, meiotic spore state (Farr et al. 2000), but Inderbitzin et al. (2006) were unable to locate a specimen with the sexual structures to serve as a type for a teleomorph name. c Hyphae (vegetative threads) that bear cells that produce macroconidia or large asexual spores. d Very small rounded mass of hyphae. e Asexual thick-walled one-cell spores. f Specialized structures that bear asci (which contain ascospores or sexual spores). In 1986, C. papaveracea/B. papaveris was evaluated as a potential biological control agent against Papaver rhoeas, a major weed of wheat in Italy (Covarelli 1981; Pignatti 1982). Results of pathogenicity and host-range experiments indicated that C. papaveracea/B. papaveris infected P. rhoeas and reduced its biomass and that it did not infect wheat, maize, barley, sorghum, or oats (Del Serrone and Annesi 1990). In 1991, researchers at the Institute of Genetics and Plant Experimental Biology in Tashkent, Uzbekistan, recovered a “highly virulent” isolate of C. papaveracea/B. papaveris from poppy plants (UNODC 2002). They reported
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104 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops that the isolate caused 50-75% losses in licit and illicit poppy crops but did not specify under what environmental conditions the losses occurred. Symptoms of disease included damping-off of seedlings and leaf and stem lesions. Poppy capsules and seeds also were affected, and this resulted in smaller capsules and reduced seed production. The discovery of the isolate became the basis of a project on the development of C. papaveracea/B. papaveris as a biological control agent against poppy (discussed in more detail later in this chapter). It is important to note that, particularly for pathogens of the foliage, environmental conditions dramatically influence the amount of damage that a pathogen can cause. Losses can approach 100% under environmental conditions favorable to the fungus, but there might be no infection or damage in conditions unfavorable to the fungus. In the late 1990s, research on the biological control of poppy (P. somniferum) was carried out at the U.S. Department of Agriculture (USDA) Agricultural Research Service (ARS) laboratory in Beltsville, Maryland, with isolates of C. papaveracea and B. papaveris recovered from poppy plants that were grown in a field in Beltsville. C. papaveracea and B. papaveris were isolated from the same diseased poppy seeds, seedlings, foliage, capsules, and field stubble and from asymptomatic plants (O’Neill et al. 2000). The two fungi are morphologically distinct, and this makes it possible to distinguish them in culture. Greenhouse and field tests conducted by Bailey et al. (2000b) showed that B. papaveris caused more severe damage on poppy than C. papaveracea. When both fungi were applied in the field, B. papaveris was the more frequently recovered fungus from poppy seed capsules and was the only fungus isolated from the field in the following year (Bailey et al. 2000b). Other studies of B. papaveris focused on whether its efficacy could be enhanced with a phytotoxic protein, Nep1, isolated from Fusarium oxysporum (Bailey et al. 2000a) or the addition of other adjuvants (Bailey et al. 2004b) and on determining the best technique for mass production of its inoculum (Bailey et al. 2004a). There have been no other publications on the evaluation or development of C. papaveracea or B. papaveris as mycoherbicides against opium poppy since the termination of the project in Uzbekistan in 2001 and the publication of the results of the studies conducted by the USDA ARS in 2000 and 2004. EFFICACY AND IMPLEMENTATION Three studies can be used as basis for assessing the efficacy of C. papaveracea or B. papaveris as a mycoherbicide agent against opium poppy. A study conducted in Italy was published in the proceedings of a conference (Del Serrone and Annesi 1990), a study conducted in the United States (Beltsville, Maryland) was published in a peer-reviewed journal (O’Neill et al. 2000), and a study conducted in Uzbekistan (UNODC 2002 report) that was sponsored by the UN International Drug Control Program under the auspices of the UN Office on Drugs and Crime (UNODC) and conducted in 1998–2002 at the Institute of
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105 Candidate Biological Control Agents against Opium Poppy Genetics and Plant Experimental Biology. The goal of the latter study was to assess the potential of C. papaveracea/B. papaveris “as an effective, reliable, and environmentally safe biological control agent for opium poppy in realistic field conditions.” The findings were presented in a report made available to the present committee; the report was reviewed by an independent expert and a group of technical experts enlisted by UNODC, but the results have not been published in peer-reviewed journals. Experiments at the Istituto Sperimentale per la Patologia Vegetale, Rome, Italy Del Serrone and Annesi (1990) conducted studies to evaluate the feasibility of controlling a species of poppy, P. rhoeas, with C. papaveracea/B. papaveris isolated from infected P. rhoeas plants in the field. P. rhoeas is an annual weed in cereal crops, especially wheat, in Italy. The committee reviewed this work because of the interest in C. papaveracea/B. papaveris as a mycoherbicide against opium poppy, although Del Serrone and Annesi did not propose to use it to control illicit poppy. Table 5-3 provides a summary of the experimental details. According to the published report, disease symptoms were observed 3-4 days after inoculation as water-soaking of petiole tissues, followed by drying of the petioles, and finally the wilting and death of leaves. The greatest damage (with over 99% of the leaves infected) was observed in plants that had four to seven true leaves and had been sprayed with a suspension of 1.5 × 106 spores/mL followed by a 24-hour dew period at 25°C. Damage (in percentage of leaves infected) was less severe when inoculated plants were given 12 hours of dew at 23°C (59%) or 24 hours of dew at 15°C (72%). The requirement for 24 hours of dew to cause the most severe damage led the authors to conclude that it is “too long” to consider the use of this isolate as a mycoherbicide. They noted that it would be useful to conduct additional investigations to find “more adaptable isolates to be used under field conditions” (Del Serrone and Annesi 1990). Experiments at the U.S. Department of Agriculture Agricultural Research Service Laboratory in Beltsville, Maryland O’Neill et al. (2000) discovered that P. somniferum seedlings and mature plants produced from USDA plant introduction seed accessions grown in greenhouses and growth chambers in Beltsville, Maryland, were dying of an unknown destructive disease. The pathogen was identified as C. papaveracea/B. papaveris. O’Neill et al. conducted replicated experiments in growth chambers to determine the pathogenicity and comparative virulence of B. papaveris (isolate B96) and C. papaveracea (isolate Cf96) to poppy plants from three
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106 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops accessions (White Cloud, Indian Grocery, and Venezuela). The details of the experiments are given in Table 5-4. The results confirmed that both fungi were pathogenic to the three poppy accessions tested, but B. papaveris B96 was generally more virulent than C. papaveracea Cf96. Symptoms of infection included chlorosis, water-soaking of the stems and leaves, and tissue death. The average disease rating (on a scale of 0-9) on all test plants 7 days after inoculation (DAI) was 8.75-9.00 (93-100% foliage necrosis) with B. papaveris, whereas the same test plants inoculated with C. papaveracea had a rating of 2.75-7.50 (6-12% to 87-93% foliage necrosis) (see Table 5-3). When a similar experiment was conducted with 18-day-old seedlings of White Cloud and Indian Grocery, the seedlings were highly susceptible to both fungi, and necrotic lesions were observed 48 hours after inoculation. Mortality was 100% in seedlings inoculated with P. papaveracea and 97% in seedlings inoculated with D. penicillatum 5 DAI. The seedlings inoculated with B. papaveris were dead, and those inoculated with C. papaveracea exhibited 87-93% necrosis. O’Neill et al. (2000) also determined the efficacy of C. papaveracea and B. papaveris in replicated experiments by inoculating poppy plants with different spore concentrations (105, 106, and 107 spores/mL) and then exposing them to different wetness periods by misting them for 0, 6, 12, 24, and 48 hours. The inoculated plants were killed within 9 days when the spore concentration was 106/mL and the wetness period was 24 hours or longer. For B. papaveris B96, at least 6 hours of wetness was required to attain 25-50% foliar necrosis 12 DAI. The efficacy of the spore inoculum increased when the inoculum was formulated with unrefined corn oil; White Cloud and Indian Grocery plants inoculated with C. papaveracea or B. papaveris spores with 30% oil and exposed to 6 hours of wetness exhibited 25-50% necrosis 3 DAI (O’Neill et al. 2000). TABLE 5-3 Greenhouse Study by Del Serrone and Annesi (1990) Factors Details Inoculum C. papaveracea/B. papaveris Spore suspension containing 1.5 × 106 spores (conidia) per milliliter with 0.001% Teepol (detergent) sprayed onto test plants until runoff; control plants sprayed with water and Teepol only Test plants Poppy plants inoculated at four growth stages: cotyledon to three true leaves, four to seven true leaves, eight to 11 true leaves, and 12-16 true leaves Environmental Inoculated and control plants exposed to different dew periods conditions (6, 12, 18, and 24 h) and temperatures (15, 20, 25, 30°C) Assessment method Efficacy assessment was based on reduction in plant dry weight and percentage of infected leaves Experiment was performed twice
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107 Candidate Biological Control Agents against Opium Poppy TABLE 5-4 Growth-Chamber Studies by O’Neill et al. (2000) Factors Details Inoculum Single-conidia isolates of C. papaveracea and B. papaveris obtained from diseased poppy foliage and capsules of plants growing in a field and growth chambers in Beltsville, Maryland Test plants Poppy plants from Indian Grocery (IG), VEN (from Venezuela, used in opium production), and White Cloud (WC) accessions Treatments In one experiment (two trials), 8-wk IG, VEN, and WC plants sprayed with B. papaveris isolate B96 and C. papaveracea isolate Cf96 spore suspensions in water (containing 4 × 104 spores/mL) and provided with 100% relative humidity (RH) for 24 h; plants then moved to a growth chamber with 40% RH, 28°C/22°C day/night temperature, and 11-h photoperiod In another experiment (two trials), 18-d-old seedlings of IG and WC sprayed with spore suspensions of isolates B96 and Cf96 in water (containing 3 × 104 spores/mL) and provided 100% RH for 24 h Different spore concentrations (105, 106, and 107 spores/mL) of isolates and different wetness periods (misting for 0, 6, 12, 24, and 48 h) also tested in growth-chamber experiments Assessment Efficacy assessed on disease-severity rating scale (based on visual method estimation of percentage of foliage blight) of 0-9 where 0 = 0-3%, 1 = 3-6%, 2 = 6-12%, 3 = 12-25%, 4 = 25-50%, 5 = 50-75%, 6 = 75-87%, 7 = 87-93%, 8 = 93-96%, and 9 = 96-100% necrosis Experiments in Uzbekistan and Tajikistan Researchers at the Institute of Genetics and Plant Experimental Biology conducted experiments in 2000-2001 to test the efficacy of fungi that they had isolated from diseased poppy plants in Uzbekistan. Their report refers to the pathogens as P. papaveracea (now referred to as C. papaveracea) and D. penicillatum (now referred to as B. papaveris) (UNODC 2002). Details of the field experiments are given in Table 5-5. In the trials in Uzbekistan, several formulations were tested, but they were not described in the report. In 2000 and 2001, application of conidia in formulation “19” resulted in reductions of 50% and 52% in poppy capsule numbers, respectively, 45% and 63% in capsule weight, and 78% and 81% in seed weight per capsule. Application of formulation “1” had similar results in the Uzbekistan trials. In the 2001 trial in Tajikistan, formulations “24,” “25,” and “27” caused the most damage: a 60% reduction in plant height, 60% in capsule numbers, and 90% in plant weight. Formulations “19” and “1,” which caused the most damage to poppies in Uzbekistan, were not as efficacious in the Tajikistan trial. The researchers thought that the higher ultraviolet-radiation levels at the Tajikistan site, which was 2,500 m above sea level, reduced the efficacy of C. papaveracea/B. papaveris formulations “19” and “1.”
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108 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops TABLE 5-5 Field Trials in Uzbekistan and Tajikistan (2000-2001) Factors Details Inoculum C. papaveracea/B. papaveris isolated from diseased opium poppy (Papaver somniferum) and wild poppies (P. arrhenium, P. pavonium, P. rhoeas, and P. refracta) in Uzbekistan and Tajikistan Isolate referred to as C-6-3 used in field experiments because it had “the greatest virulence and highest growth rate” Location Trials conducted in Uzbekistan and Tajikistan field sites and time of Inoculations in autumn, at “budding phase” of poppy plants inoculation Inoculum suspension containing 1 × 106 conidia/mL sprayed at 500 L/ha Treatments (5 × 1011 conidia/ha); treatments consisted of spores in different formulations identified by numerical designations but of undescribed compositions; control treatment consisted only of formulation without spores Assessment Efficacy of fungus assessed on basis of reduction in poppy capsule numbers, method capsule weight, and weight of seeds per capsule in Uzbekistan trials; in Tajikistan trials, efficacy assessed on basis of reduction in plant height, capsule numbers, and plant weight Final yield data not obtained, because “it was too dangerous to keep the trial long enough for this” According to the Uzbek researchers, inoculation of poppy plants at the rosette stage resulted in leaf infections followed by stem infections and finally plant death within 48 hours of inoculation. Infection at a more mature, capsule- forming stage resulted only in capsule discoloration and stunting. The re- searchers further reported that under natural field conditions, C. papaveracea or B. papaveris can cause disease in plants at the “budding phase” (capsule formation) and that although inoculation at this stage did not cause plant death, the capsule was so affected that it was commercially unusable; capsules of infected plants were small, hard, and blackened and contained seeds with lower viability. Laboratory analysis for alkaloids showed reduced concentrations of morphine, codeine, thebaine, narcotine, and papaverine in plants that were inoculated with fungal formulations “1” and “19” (UNODC 2002). The authors recommended application of the fungus to poppy plants at the rosette stage (before flowering) to destroy the crop (UNODC 2002). Del Serrone and Annesi (1990) demonstrated that younger plants are more susceptible to damage and that the fungus requires 24 hours of dew to cause severe damage. O’Neill et al. (2000) also showed that young plants are more susceptible to damage and that C. papaveracea or B. papaveris can cause severe damage or death relatively quickly if the inoculated plants are exposed to a long wetness period, that is, at least 24 hours. However, in a study by Bailey et al. (2000b), field-grown poppy plants from the accession White Cloud inoculated at the rosette stage did not become severely infected until the plants began to flower and form capsules. Bailey et al. postulated that the susceptibility of younger plants in greenhouse experiments was due to the optimal conditions in
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109 Candidate Biological Control Agents against Opium Poppy the greenhouse, which may not always occur in the field. An important caveat here is that the different research groups worked with different isolates of C. papaveracea or B. papaveris (see Table 5-1). Results from greenhouse and field studies provide evidence that C. papaveracea and B. papaveris can cause disease on poppy, but the extent of damage or yield loss will depend on several factors, including the virulence of the isolate or strain used, the inoculum formulation, the application rate, the plant growth stage, the poppy cultivar (genotype), and the environmental conditions in the field during and immediately after inoculation. Mechanisms of Pathogenicity Pathogenicity is the ability to cause disease; disease may result from a pathogen’s infecting, colonizing, and disrupting the normal cellular functions of a plant. C. papaveracea and B. papaveris infect aerial parts of the plant, initially causing chlorotic spots on the leaf, chlorosis on leaf margins, and water-soaking on the leaf and stem, which may be followed by withering and drying of the leaf and the development of stem lesions (Milatović 1975b; Del Serrone and Annesi 1990; Bailey et al. 2000b; O’Neill et al. 2000). Infected leaves change from green to gray-green and enter senescence prematurely (Miltović 1975b). Both C. papaveracea and B. papaveris form appressoria, specialized suction-cup-like structures that some fungi require to penetrate plant epidermal cells and cause infection, on poppy leaves, but they can also penetrate the leaf through open stomata (Bailey et al. 2000b). The disease can attack any part of the opium poppy plant—leaf, stem, capsule, root, or seed—at any stage of development (Milatović 1975a). Although diseased seedlings and immature plants might be killed (Del Serrone and Annesi 1990; Bailey et al. 2000b; O’Neill et al. 2000), premature drying of the plant at any of the later stages of development (flowering, capsule formation, or capsule maturity) can greatly reduce the number, size, and weight of the capsules (Bailey et al. 2000b) and the amount of opium that could be harvested from the still-green capsules (UNODC 2010d). C. papaveracea produces sexual and asexual spores on infected poppy tissues (Milatović 1975b; O’Neill et al. 2000); these spores may disperse the pathogen throughout the field and region. Being a seedborne pathogen (Milatović 1975b), C. papaveracea also could be spread through the use of infected seed for planting. Little is known about the mechanisms underlying the pathogenicity of C. papaveracea or B. papaveris. As noted in Chapter 4, filamentous fungi produce a variety of secondary metabolites (organic compounds produced by an organism that are not required for its immediate survival). Although the function of most secondary metabolites is unknown, many have been shown to have biological activity, including phytotoxicity. The UNODC (2002) report listed several metabolites produced by C. papaveracea that were identified in methanol extracts of fungal cultures and that were found to be phytotoxic in a
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110 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops detached-leaf assay. Phytotoxicity was measured semiquantitatively (+, ++, +++) over a 48-hour period. Phytotoxins identified included 1, 2-benzene dicarboxylic acid; 1, 2-benzene dicarboxylic acid, dipropyl ester; 1, 2-benzene dicarboxylic acid, bis (ethylhexyl) ester; and two derivatives of tetracosahexaene (squalene). These metabolites have no clear role in disease development (UNODC 2002). Some experiments combining a culture filtrate with the fungus were performed to determine whether fungal efficacy was enhanced by toxic metabolites; the results showed little enhancement of the efficacy of C. papaveracea (UNODC 2002). The role of the metabolites in the pathogenicity of C. papaveracea, if any, remains unknown. INOCULUM PRODUCTION AND DELIVERY Facility, Equipment, and Technology for Large-Scale Manufacture A general discussion of the fermentation methods used for large-scale production of commercial mycoherbicides is provided in Chapter 4 (see section “Inoculum Production and Delivery”). C. papaveracea/B. papaveris can be grown to produce infective propagules on a variety of solid substrates and liquid media (Table 5-6). On the basis of studies reviewed in Table 5-6 and the general literature on microbial pesticides, it appears feasible to mass-produce these fungi. The available data on the proposed mycoherbicides provide useful leads but are exploratory; any large-scale attempt at production would be more efficient if it begins with basic studies of fungal growth and fermentation, preferably in collaboration with an industrial partner. Such studies are needed to determine the choice of fermentation method (liquid, solid, or biphasic), the type of propagule (mycelium, conidium, chlamydospores, microsclerotia, pseudo- thecia, or ascospores), the formulation (liquid concentrate, dust, pellet, food- grain-based, or seed-based), the intended delivery method (aerial or ground- based), and the expected efficacy, shelf-life, and handling qualities of the product. To estimate the amounts of the mycoherbicide product that might be required for a hypothetical program to control illicit opium poppy worldwide, the committee made calculations on the basis of published data on the amounts of inoculum used in field trials (Table 5-7). However, as noted in Chapter 4, the published data are only a guide; the actual amounts cannot be determined without testing the finished mycoherbicide product under conditions that simulate operational programs. Typically, this phase of mycoherbicide research and development is done by an industrial producer in collaboration with the mycoherbicide researchers. Therefore, the actual amounts of opium-poppy mycoherbicide required may be higher or lower than the amounts projected in the table. The estimates in Table 5-7 project that large volumes of water would be necessary to apply the opium-poppy inoculum, and this could be an important limiting factor in developing it as a mycoherbicide.
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TABLE 5-6 Methods Used for Production of C. papaveracea/B. papaveris Inoculum for Experimental Trials Reference Method Inoculum Produced Del Serrone and Inoculum produced by culturing C. papaveracea/ Spores produced (type not mentioned; presumed to be conidia) Anessi 1990 B. papaveris on malt extract agar (30% maltose) for used for inoculation 9 days at 25°C with 12-h light and dark periods Bailey et al. 2000b; C. papaveracea/B. papaveris cultured on V8 agar medium Conidia from agar plate cultures used in laboratory, greenhouse, O’Neill et al. 2000 for 7-10 days at 23°C with 16-h photoperiod and field experiments Bailey et al. 20004b C. papaveracea and B. papaveris cultured on V8 agar Conidia from agar plate cultures used in laboratory, greenhouse, medium for 6-10 days at 25°C in the dark and field experiments Bailey et al. 2004a C. papaveracea/B. papaveris cultured on agar with Greatest amount of radial growth occurred on molasses-brewer’s molasses, wheat bran, pectin, rice flour, dextrin, yeast, soy fiber-brewer’s yeast, and wheat bran-brewer’s yeast agar cornstarch, soy flour, corncobs, or cottonseed meal, media; greatest number of conidia produced on soy fiber-brewer’s each with brewer’s yeast yeast agar medium; only chlamydospores produced on cornstarch- brewer’s yeast and dextrin-brewer’s yeast agar media 1 × 106 colony forming units per milliliter produced on all four Bailey et al. 2004a Liquid media (in 100-mL shake flasks) containing dextrin, substrates after 5 days of growth; chlamydospores (6 × 105/mL) cornstarch, wheat bran, or soy fiber, each supplemented with brewer’s yeast produced only in media containing cornstarch-brewer’s yeast and dextrin-brewer’s yeast; conidia (104/mL) produced only in media containing soy fiber-brewer’s yeast and wheat bran-brewer’s yeast Bailey et al. 2004a Bench-top fermentation (2.5-L commercial bench-top Biomass produced on bench-top fermentor consisted of fermentor) with dextrin (30.0 g) and brewer’s yeast (15.0 g) nonmelanized mycelial fragments and chlamydospores mixture as medium; medium maintained at 25°C with 200- rpm agitation, fermentation period was 7 days UNODC 2002 10-mL conidial suspension of C. papaveracea added to Dry spores stored in sealed or nonsealed containers or mineral oil; 250 mL of liquid medium in flask; inoculated liquid medium tests conducted over a 5-year period indicated minimal reduction (not identified in report) incubated on rotary shaker (70 rpm) in spore viability and virulence; number of propagules produced at room temperature for 3-4 days, after which mycelia were not discussed in report recovered and spread out on muslin cloth that had been stretched over metal frame; mycelia on muslin incubated at 100% relative humidity for 48 h; spores then collected with vacuum harvester and stored as dry powder 111
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112 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops TABLE 5-7 Estimated Amounts of the Proposed C. papaveracea/B. papaveris Needed for a Single Application against Illicit Opium-Poppy Crops Worldwide Amount of Mycoherbicide (No. Spores) and Volume Amount of Mycoherbicide Amount of Inoculum and of Water Needed for Each (No. Spores) and Volume of Volume of Carrier (Water) Water Needed per Hectare Application over Reference Used in Field Trialsa Worldwide Areab,c,d for Each Application Bailey et 2 × 105 conidia/mL 6.5 × 1015 to 13 × 1015 3.6 × 1010 to 7.2 × 1010 al. 2000b suspended in Tween 20 (6.5-13 quadrillion) spores and 182-364 L or Tween 20 plus corn oil, spores and 33-66 million of water applied at 3 mL/Plant or liters of water 180-360 L/hae Bailey et 5 × 105 conidia/mL 1.2 × 1017 (1.2 sextillion) 6 × 1011 spores and al. 2000ae suspended in water or spores and 234 million 1,290 L of water aqueous adjuvant, applied liters of water at 5 × 1010 conidia/ha in 1,290 L of water (with or without adjuvants) Bailey et 1 × 106 /mL conidia 1.6 × 1017 (1.6 sextillion) 88 × 1010 spores and al. 2004be applied at 8.7 × 1011 spores and 157 million 866 L of water conidia/ha in 866 L/ha liters of water 331 × 1010 spores and 6 17 1 × 10 /mL conidia 6 × 10 (6 sextillion) 3,303 L of water applied at 3.3 × 1012 spores and 599 million 992 × 1010 spores and conidia/ha in 3,300 L/ha liters of water 4,951 L of water 2 × 106 /mL conidia 1.8 × 1018 (1.8 quintillion) applied at 9.9 × 1012 spores and 898 million conidia/ha in 4,950 L/ha liters of water 5 g/0.3 m2 of inoculum 90 × 1015 (90 quadrillion) 50 × 1010 spores and UNODC 2002 composed of fungus- spores and 91 million 502 L of water colonized millet seeds; liters of water proportion of fungus to millet seed not specified a Amounts of inoculum used in field experiments were not aimed at defining the minimum inoculum quantity needed for effectiveness; this remains to be determined with the actual mycoherbicide product. b Based on most recent UNODC estimate of total area under opium-poppy cultivation worldwide is 181,373 ha (UNODC 2010a). These calculations are provided on the basis of a potential worldwide target area to indicate the industrial production capacity that might be needed. The committee regards simultaneous worldwide application or even a worldwide application within a growing season as logistically unrealistic. c A typical opium-poppy field has 60,000-120,000 plants/ha (see Chapter 3). d Without knowing the weight of each spore, it is not possible to estimate the required amount in metric tons. e Descriptions of plot size are incomplete. Estimation is based on the committee’s interpretation of the available details.
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113 Candidate Biological Control Agents against Opium Poppy Adjuvants and Formulation For a general discussion of adjuvants and formulations, see the section “Adjuvants and Formulation in Chapter 4. The use of adjuvants to improve the efficacy of C. papaveracea and B. papaveris against opium poppy was studied by Bailey et al. (2000b, 2004b) and by O’Neill et al. (2000). Among the adjuvants tested, the most effective were Tween 20 (polyoxyethylene  sorbitan monolaurate) and unrefined corn oil. The details and results of the experiments are summarized in Table 5-8. O’Neill et al. (2000) showed that the addition of unrefined corn oil to C. papaveracea and B. papaveris inoculum increased the severity of disease caused by these two fungi in White Cloud and Indian Grocery poppy plants. They noted that the formulation of C. papaveracea spores with corn oil and the provision of a 6-hour wetness period rendered C. papaveracea “almost as virulent” as B. papaveris on the tested poppy cultivars. Researchers in the Institute of Genetics and Plant Experimental Biology also tested various formulations of C. papaveracea/B. papaveris under field conditions, but they identified the formulations only by number and did not provide any information on their composition (UNODC 2002). The use of a low concentration of Tween 20 as an adjuvant might be cost- effective, but the environmental effects of its use might require documentation for registration purposes. The use of unrefined corn oil at 10-30% by volume of the spray mixture is impractical in light of the large spray volumes required (Table 5-6). TABLE 5-8 Effect of Adjuvants on the Efficacy of C. papaveracea/B. papaveris in Greenhouse and Field Experiments Reference Adjuvant Tested Efficacy In greenhouse experiments, application of spores (106/mL) Bailey et al. 0.001% Tween 20 2000b in 10% (unrefined) mixed with 0.001% Tween 20 in 10% corn oil resulted in corn oil 100% infection by B. papaveris and nearly 100% infection by C. papaveracea; B. papaveris with 0.001% Tween 20 in 10% corn oil caused 57.7% mortality in poppy seedlings; C. papaveracea with 0.001% Tween 20 in 10% corn oil caused 34.7% mortality in poppy seedlings; and 17% of plants sprayed with spores of either pathogen with 0.0001% Tween 20 in water did not exhibit any disease symptoms O’Neill et al. 30% (unrefined) In greenhouse tests, poppy plants inoculated with C. 2000 corn oil papaveracea and B. papaveris spores mixed with 30% corn oil and provided with a 6-hour wetness period had 25-50% foliar necrosis at 3 DAI Bailey et al. 1% Tween 20 In field experiments, B. papaveris spores mixed with 1% 2004b Tween 20 caused 68% and 56% necrosis and 22% and 27% reduction in capsule weight per plot within 2 weeks of application in trials 1 and 2, respectively
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114 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops Delivery On-ground application of a mycoherbicide against opium poppy is possible, but from a tactical standpoint, aerial application may be the only feasible delivery method over inaccessible areas. An aerial pathogen such as C. papaveracea/B. papaveris is likely to be most effective when sprayed on the plants (that is, aerial application). However, all the studies that the present committee reviewed have examined only land-based spray application of C. papaveracea/B. papaveris directed at the plants. Of the five papers that the committee reviewed (Bailey et al. 2000a,b, 2004b; O’Neill et al. 2000; UNODC 2002), two identified the tool used to apply the inoculum (Bailey et al. 2000a, 2004b), namely, a Binks spray gun model #15 delivering a spore suspension at 15 lb/in2. Presumably, the other studies used some type of hand-held or backpack sprayer. Aerial spraying of a liquid formulation of C. papaveracea/B. papaveris is unlikely to be practical, because of the large quantities of water that would be required if the methods used by the researchers were implemented on a large scale (see Table 5-6). Producing the large number of spores required might not constitute a problem, provided that a suitable fermentation method is found, but the availability of water and the ability to transport and apply the required quantities in the field would pose major challenges. Assessment of Performance The opium-poppy crop is grown in open fields under full sun, so it should be possible to assess the performance of the C. papaveracea/B. papaveris mycoherbicide with aerial imagery. Although the technology for aerial imagery is available, it has to be adapted and tested for use in measuring mycoherbicide efficacy. As an alternative, on-ground assessment of crop damage combined with interviews with growers could be used; however, this approach requires the ability to ensure personnel security and access to targeted regions. The assessment methods used by the researchers were not developed for use in operational drug-crop control programs. For example, in the field trials conducted in Uzbekistan, the efficacy of C. papaveracea/B. papaveris was assessed on the basis of reductions in poppy capsule numbers, capsule weight, and weight of seeds per capsule. In the Tajikistan trials, efficacy assessment was based on reductions in plant height, capsule numbers, and plant weight. Final yield data were not obtained in these studies, because it was too “dangerous” to continue the trial (UNODC 2002). O’Neill et al. (2000) assessed the efficacy of C. papaveracea and B. papaveris in growth-chamber studies with a disease- severity rating scale (see Table 5-4). Del Serrone and Annesi (1990) assessed the efficacy of C. papaveracea/B. papaveris on the basis of plant dry-weight reduction and percentage of infected leaves.
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115 Candidate Biological Control Agents against Opium Poppy PERSISTENCE IN THE ENVIRONMENT The persistence of C. papaveracea/B. papaveris in the environment is an important factor in determining whether the applied fungal strain(s) could potentially affect later plantings of the crop. If the mycoherbicide poses risks to nontarget organisms after release, its prolonged persistence would be a disad- vantage rather than an advantage. Thus, as noted in Chapter 4, an understanding of the ability to persist in the environment is an important consideration in proposing the use of a mycoherbicide. Geographic and Climatic Considerations Only a few studies provide quantitative information on the survival of C. papaveracea/B. papaveris (Del Serrone and Annesi 1990; Bailey et al. 2000b; UNODC 2002). Del Serrone and Annesi (1990) found that moisture on the plant surface for sufficiently long durations (length of time depending on the temperature) at favorable temperatures (10-30°C) are required for spore germination and later penetration of host tissue. All the studies were done at 100% humidity or on wet leaflets. Bailey et al. (2000b) performed a detached-leaf assay with C. papaveracea and B. papaveris to assess the effect of temperature on their survival and growth on poppy leaf surfaces. Survival and growth were measured in terms of conidial germination, germ-tube growth, and formation of appressoria at various temperatures. Germ-tube growth and appressorium formation were favored at higher temperatures (16-29°C), whereas conidial germination was similar throughout the range of temperatures tested (7-29°C). B. papaveris formed more appressoria than C. papaveracea regardless of temperature and required fewer hours of moisture. The long-term survival of C. papaveracea and B. papaveris also was assessed as part of field tests of their infectivity (discussed above in the section “Efficacy and Implementation”). After tests during the first season were completed, the remaining poppy plant residues in the field were chopped and left on the soil surface over the winter. In the spring, opium poppies were planted in the fields, and plants that had symptoms of disease were tested for the presence of the fungi. Only B. papaveris was isolated from diseased poppy. The investigators suggested that their results could be due to this pathogen’s ability to produce ascospores in the spring (Bailey et al. 2000b). In the studies in Uzbekistan (UNODC 2002), laboratory and field tests indicated that conidia of C. papaveracea/B. papaveris required the presence of the host plant to survive for longer than 2-3 months. In the presence of host tissue, the pathogen remained viable for up to 15 months when applied to the top 5 cm of soil and for up to 6 months when applied at a depth of 15 cm. In the absence of the host plant, persistence was reduced to 3 months at all depths
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116 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops during the winter and to 2 months at all depths during the summer. When plant debris was buried in the soil, viable fungi were detected for up to 10 months. However, the UNODC report provided no description of the testing protocols or analytical methods that were used. As previously stated, such physical factors as unfavorable temperature, moisture, and solar radiation and such biological factors as antagonistic microorganisms in the soil could influence survival of C. papaveracea and B. papaveris, but there are no specific data on the effects of these factors on the two pathogens. Inasmuch as C. papaveracea and B. papaveris already are present in most areas where poppies are grown, the environmental conditions and the life cycle of the plants seem to be such that at least some level of persistence would be achieved. Transmission and Spread The potential pathways by which spores and vegetative propagules from a particular application site might move into environmental media are illustrated in Chapter 2 (see Figure 2-1). In general, dispersal of the proposed mycoherbicides after application would depend on the production and natural dispersal of secondary inoculum. Experience with other plant pathogens indicates that short-distance spread of ascospores is facilitated by rain and wind, whereas dispersal of conidia occurs primarily by wind (Li and Kendrick 1995; Hildebrand 2002). Long-distance transport and dispersal by wind and water are possible for infected seeds, infected plant tissues, infested dead plant material, and conidia (Meffert 1950; UNODC 2002). It is also possible for farmers and traders to carry infected or infested materials throughout a region or even into new areas. Other Considerations The occurrence and susceptibility of the host plant appears to be a major factor in determining the population size of many pathogens, including C. papaveracea and B. papaveris. The pathogens are somewhat host-specific, being capable of infecting P. somniferum and other species of Papaver (Del Serrone and Annesi 1990; UNODC 2002). The pathogens might survive on related hosts or colonize unrelated hosts that are not necessarily susceptible to them, as has been shown with formae speciales of Fusarium oxysporum (see Chapter 4), but there are no data on the presence of C. papaveracea and B. papaveris on or in tissues of nontarget plants. These fungi are widely prevalent across the range where opium poppy is grown (Schmitt and Lipscomb 1975). No studies are available on the interactions of C. papaveracea or B. papaveris with soil microorganisms or other organisms. In general, as noted in Chapter 4, the presence of competitor or antagonistic microorganisms could reduce the persistence C. papaveracea or B. papaveris. For example, insects and
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117 Candidate Biological Control Agents against Opium Poppy soil organisms can feed on or suppress plant-pathogenic fungi (Nakamura et al. 1992; Okabe 1993; Suárez-Estrella et al. 2007). Thus, antagonistic micro- organisms in the soil could theoretically reduce the likelihood that a C. papaveracea/B. papaveris mycoherbicide would establish populations that are large enough to cause recurrent disease in opium poppy. It is possible that the introduced strains of C. papaveracea or B. papaveris could displace the resident strains; but there are no data available to determine the probability of such displacement or its consequences. Overall, the ecological requirements for the spread and survival of C. papaveracea and B. papaveris cannot be adequately described on the basis of the studies conducted so far. In general, it appears that the pathogens could persist in soil for at least two growing periods in the presence of host tissue. The fact that they are found almost everywhere that opium poppy is grown indicates that the strains distributed are likely to persist at some level once introduced into a site. Although no data are available specifically on the potential dispersal of the fungi from the site of application, it is reasonable to assume that they would spread through infected or infested seeds, plant tissues, or plant debris and by wind and rain. EFFECTS ON NONTARGET ORGANISMS Microbial pesticides are regulated by the U.S. Environmental Protection Agency (EPA), which requires a variety of tests on the environmental fate and safety of pesticides before they are registered. Chapter 2 and Appendix B describe the types of testing required—including product analysis, pesticide- residue analysis, toxicity testing, and toxicity and pathogenicity testing of nontarget organisms—and the assessment of environmental fate. The afore- mentioned studies have not been systematically performed for the EPA registration of C. papaveracea/B. papaveris. This section reviews the data avail- able on C. papaveracea/B. papaveris in the open literature that are pertinent to an understanding of their potential adverse effects on nontarget plants, animals, and microorganisms. Effects on Nontarget Plants or Microorganisms In the UNODC (2002) study, the host specificity of C. papaveracea/B. papaveris was tested against 239 species of plants of economic, medicinal, and ornamental importance, including trees and shrubs, and 52 wild species of plants belonging to 24 families. Some nontarget Papaver species were reported to be susceptible, but none of the other nontarget plants was susceptible. The report provides no names of the plant species and families tested, including the names or numbers of the species in Papaveraceae, and no details of the testing and assessment methods. Hence, the information available cannot be used to assess the risks posed to nontarget relatives of poppy by C. papaveracea/B. papaveris.
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118 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops Del Serrone and Annesi (1990) tested 14- to 20-day-old plants of “several varieties” of cereal crops and three Papaver species by spraying them with “the optimum” suspension of mitospores (conidia) of C. papaveracea/B. papaveris and growing the plants under “the best conditions for disease development.” “Poppy” plants were included as a control. None of the cereal-crop varieties tested developed disease by 14 DAI, and the fungus was never reisolated from them. Papaver dubium and P. nudicaule, a wild and a cultivated species, respectively, developed a hypersensitive (resistant) reaction. A few small, black, round spots appeared on the surface of the leaves. Some 30% of the P. somniferum plants had died at 5 DAI. The fungus was not recovered from P. dubium, P. nudicale, and P. somniferum, so there was no evidence that the fungus caused the disease. Effects on Legal Crop Production The potential risk to legal production of poppy has not been given any attention in the literature reviewed by the committee. Licit poppy crops may be cultivated for seed, oil, ornamental uses, and pharmaceutical purposes to extract narcotics. It is possible that C. papaveracea/B. papaveris would be present in fields used for legal production. If legal poppy production occurs in or near illicit-opium-producing regions, the inundative release of C. papaveracea/B. papaveris as a mycoherbicide to control the illicit plants could similarly enhance the development of disease in the legal crop owing to drift during application or by secondary inoculum produced on infected plant tissues. Toxicity to Wildlife, Domestic Animals, and Humans Although the UNODC (2002) report described secondary metabolites of C. papaveracea/B. papaveris that are phytotoxic, none of the metabolites was tested for mycotoxigenic activity. Two secondary metabolites were identified as derivatives of tetracosahexaene (squalene). Why the authors described the squalene derivatives as “fumonisin-like” is not clear. Squalene and fumonisins are not derived from the same biosynthetic pathway, so there is no rationale, on the basis of the spectral analyses (mass, infrared, and ultraviolet spectrometry), to conclude that the derivatives are fumonisin-like, and the conclusion that C. papaveracea produces fumonisin mycotoxins is not well supported. The committee found no publications other than the UNODC report on biologically active metabolites, including mycotoxins, produced by C. papaveracea/B. papaveris. Pathogenicity in Animals and Humans No reports of human or animal infection with Pleospora, Crivellia, or
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119 Candidate Biological Control Agents against Opium Poppy Brachycladium were found. A single report on the toxicity of culture extracts of Pleospora for cell lines was reviewed (Ge et al. 2005), but it was not helpful in evaluating the potential risks of infection of humans or animals. If those fungi are shown to be thermotolerant (that is, able to grow efficiently at human body temperature, 37°C), there would be a theoretical risk that increasing their amounts in the environment might lead to infection in immunocompromised humans and animals. However, on the basis of the absence of any case reports, the likelihood appears quite low. MUTATION The potential for mycoherbicides to mutate is similar to that of fungi in general, as described in Chapter 4. The diversity of fungal genotypes also is affected by sexual recombination within species. Many fungi that have not been observed to reproduce sexually may do so cryptically, judging from population- genetics evidence (Taylor et al. 2000). In some cases, population-genetics evidence on sex has led to confirmation by laboratory mating (O’Gorman et al. 2009). New genetic variation can become established in fungal populations by natural selection or by chance. Adaptation to new environments, for example, to new plant hosts or to new cultivars of crop plants can be accelerated by outbreeding and recombination due to sexual reproduction (Goddard et al. 2005; Zhan et al. 2007; Sommerhalder et al. 2010). All those processes could affect Crivellia or Brachycladium species. However, there is little basic genetic information on C. papaveracea or B. papaveris, so only a generalization about the potential for mutation can be made. There is no reason to expect that the mutation rate of these fungi would be different from that of other filamentous fungi or that they would be more or less susceptible to gene gain, gene duplication, or horizontal gene transfer. C. papaveracea outbreeds by sexual reproduction. B. papaveris reproduces sexually but is homothallic (self-mating) and need not produce recombined progeny (Farr et al. 2000). Thus, adaptation involving virulence or host range could be accelerated by genetic recombination in the case of C. papaveracea but not necessarily in the case of B. papaveris. Mutation could play a role in determining the toxicity (with respect to secondary metabolites produced) of a mycoherbicide to the extent that mutation results in changes in the amount of toxin produced or the environmental conditions under which the toxins are produced. Concerns about mutation- related changes in the toxicity of C. papaveracea or B. papaveris are all but impossible to assess because very little research has been performed. As noted earlier, the available data are insufficient to determine what secondary metabolites are produced by C. papaveracea or B. papaveris, let alone in what quantities or how production would be affected by mutations.
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120 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops A PRESUMPTIVE DISEASE EPIDEMIC IN OPIUM POPPY IN AFGHANISTAN According to UNODC, diseases of opium poppy are a normal occurrence in Afghanistan. Farmers report various degrees of damage to their crops in practically all years and regions since UNODC began systematic yield surveys (UNODC 2010d). In the spring of 2010, a fungal disease was speculated to be the possible cause of an opium-poppy blight in Afghanistan. The poppy plants exhibited wilting and other disease symptoms that appeared to be consistent with a fungal infection. Tests of diseased tissues identified two Fusarium species, but they were probably secondary colonizers of the decaying tissue rather than the cause of the disease. C. papaveracea/B. papaveris, which has been linked with past diseases of opium poppy in Afghanistan, was not detected (personal communication, Justice Tetty, UNODC, November 19, 2010), but it was noted that the tissue samples examined were of poor quality (personal communication, Eric Boa, CABI, November 25, 2010). The UN Afghanistan Opium Survey of 2010 reported that opium production was 48% lower than in 2009 (UNODC 2010d) although the overall area under poppy cultivation remained the same. Disease was considered a major contributor to the reduction in opium yield, but farmers also reported losses due to frost, drought, and pests, such as aphids, other insects, and worms. Poppy capsules were fewer and smaller than in previous years. It is important to note that diseases in major growing areas affected opium poppy plants at the late stage of plant development. The diseased plants were described as exhibiting wilt symptoms with yellowing of leaves, drooping, and finally desiccating completely, all of which are indicative of a collar (stem-root interface) or upper root rot. Those symptoms are consistent with the ones observed previously in the region in connection with fungal infections (UNODC 2010d) but are inconsistent with the typical symptoms of infection by C. papaveracea/B. papaveris. The southern region was the most affected: about 42% of the area under opium cultivation was damaged. The western region was also affected by diseases but to a much smaller degree. In the west, a combination of factors, including frost, played a role, according to farmer reports (UNODC 2010d). On the basis of the foregoing account, the cause of the reduction in opium production in 2010 in Afghanistan is unknown. Diseases, drought, frost, and pests might have contributed to it. Adverse weather conditions (such as frost and drought) might have predisposed the 2010 crop in different parts of Afghanistan to diseases. Without conclusive evidence based on positive identification of the pathogen, C. papaveracea/B. papaveris could not be implicated in the 2010 Afghan poppy blight epidemic. Therefore, it is not possible to gain any insight from this epidemic to guide the use of C. papaveracea/B. papaveris as a mycoherbicide.