4

Fusarium oxysporum formae speciales as Candidate Biological Control Agents for Cannabis and Coca

The genus Fusarium is one of the largest genera of fungi and includes species that reproduce clonally and by recombination and species that reproduce only clonally (Leslie and Summerell 2006). F. oxysporum is a species complex (O’Donnell et al. 2004) that is pathogenic to a wide variety of plant species, including several economically important vegetable and ornamental crops (Nelson et al. 1981; Michielse and Rep 2009). Sexual reproduction by F. oxysporum has not been observed. However, genetic variation is significant in populations of nonpathogenic strains, and strains of some form species (forma specialis, abbreviated f.sp.; plural formae speciales, abbreviated f.spp.) have few clonal genotypes and large amounts of genetic variation (Leslie and Summerell 2006). As Baayen et al. (2000) state, “although a teleomorph has not yet been found, the sexual cycle may still be active in FOC [F. oxysporum complex].” F. oxysporum occurs in all types of soils worldwide and causes severe vascular wilts, damping-off, and crown and root rot in its hosts (Jarvis and Shoemaker 1978; Nelson et al. 1981; Summerell et al. 2001; Di Pietro et al. 2003). In the absence of a plant host, it can exist as a saprophyte (an organism that lives on dead organic matter) in soil for extended periods (Burgess 1981).

F. oxysporum has at least three and probably more species-level clades (O’Donnell et al. 2004). Isolates are placed in form species on the basis of the host-plant species that is attacked (Armstrong and Armstrong 1981; Kistler 1997). More than 150 form species of F. oxysporum have been characterized (Armstrong and Armstrong 1981; Baayen et al. 2000). Nonpathogenic strains of F. oxysporum also exist in soil and are distinguished from pathogenic strains through pathogenicity testing (Edel et al. 2001). In some instances, a F. oxysporum form species can have a wide host range and a single genotype is capable of parasitizing several different plants. For example, F. oxysporum f.sp. vasinfectum can be isolated from cotton, alfalfa, and tobacco (Assigbetse et al.



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4 Fusarium oxysporum formae speciales as Candidate Biological Control Agents for Cannabis and Coca The genus Fusarium is one of the largest genera of fungi and includes species that reproduce clonally and by recombination and species that reproduce only clonally (Leslie and Summerell 2006). F. oxysporum is a species complex (O’Donnell et al. 2004) that is pathogenic to a wide variety of plant species, including several economically important vegetable and ornamental crops (Nel- son et al. 1981; Michielse and Rep 2009). Sexual reproduction by F. oxysporum has not been observed. However, genetic variation is significant in populations of nonpathogenic strains, and strains of some form species (forma specialis, ab- breviated f.sp.; plural formae speciales, abbreviated f.spp.) have few clonal genotypes and large amounts of genetic variation (Leslie and Summerell 2006). As Baayen et al. (2000) state, “although a teleomorph has not yet been found, the sexual cycle may still be active in FOC [F. oxysporum complex].” F. ox- ysporum occurs in all types of soils worldwide and causes severe vascular wilts, damping-off, and crown and root rot in its hosts (Jarvis and Shoemaker 1978; Nelson et al. 1981; Summerell et al. 2001; Di Pietro et al. 2003). In the absence of a plant host, it can exist as a saprophyte (an organism that lives on dead or- ganic matter) in soil for extended periods (Burgess 1981). F. oxysporum has at least three and probably more species-level clades (O’Donnell et al. 2004). Isolates are placed in form species on the basis of the host-plant species that is attacked (Armstrong and Armstrong 1981; Kistler 1997). More than 150 form species of F. oxysporum have been characterized (Armstrong and Armstrong 1981; Baayen et al. 2000). Nonpathogenic strains of F. oxysporum also exist in soil and are distinguished from pathogenic strains through pathogenicity testing (Edel et al. 2001). In some instances, a F. ox- ysporum form species can have a wide host range and a single genotype is capa- ble of parasitizing several different plants. For example, F. oxysporum f.sp. va- sinfectum can be isolated from cotton, alfalfa, and tobacco (Assigbetse et al. 61

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62 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops 1994). Other forms show evidence of convergent evolution: several genotypes have independently evolved the ability to parasitize the same plant species, for example, F. oxysporum f.sp. cubense (O’Donnell et al. 1998). BACKGROUND ON THE SPECIFIC FUNGI Biological Control of Cannabis with Fusarium oxysporum f.sp. cannabis Plants in the genus Cannabis, including C. sativa (fiber hemp and canna- bis), are attacked by a number of pathogens, some of which may cause serious damage or plant death. A review of scientific literature on cannabis pathogens by McPartland (1992) identified at least 88 fungal species that attack cannabis. Among those fungi are several Fusarium species that cause damping off, stem canker, root rot, and wilt (McPartland 1996). Fusarium wilt of hemp, caused by F. oxysporum f.sp. cannabis and F. oxysporum f.sp. vasinfectum, was first noted in eastern Europe (Russia, the Czech Republic, Poland, and Romania) more than 50 years ago; it also reportedly occurs in western Europe (Italy and France), central Asia (Kazakhstan and Pakistan), and North America (Canada and the United States) (McPartland and Hillig 2004). F. oxysporum f.sp. cannabis and F. oxysporum f.sp. vasinfectum are mor- phologically similar in culture, but, according to McPartland and Hillig (2004), the two can be differentiated on the basis of their host range. F. oxysporum f.sp. cannabis is reported to infect only cannabis, whereas the host range of F. ox- ysporum f.sp. vasinfectum includes (in addition to cannabis) cotton, mungbean, pigeon pea, rubber tree, alfalfa, soybean, coffee, tobacco, and other plants (McPartland and Hillig 2004). The study of genetic variability with DNA poly- morphisms may provide an alternative route for identification of these two for- mae speciales. F. oxysporum f.sp. cannabis was considered a potential control agent for cannabis as early as the 1970s on the basis of its purported specificity to mem- bers of the genus Cannabis, its ability to survive in the soil for extended periods, and the likelihood of infecting new plantings of the crop. Hildebrand and McCain (1978) conducted laboratory experiments to develop a suitable tech- nique for the production of F. oxysporum f.sp. cannabis inoculum consisting of chlamydospores, which are suitable for soil application. Later, McCain and No- viello (1985) explored the feasibility of using F. oxysporum f.sp. cannabis (iso- lated in Italy) as a biological control agent against C. sativa and claimed that the fungus caused disease only on C. sativa and that it was able to survive in the soil for at least one growing season. In the late 1990s, Tiourebaev et al. (2001) conducted experiments to test the pathogenicity of F. oxysporum f.sp. cannabis isolates obtained from diseased cannabis plants collected in various regions of Kazakhstan and to determine their virulence, their host range, and the formulation best suited for field applica- tion. The findings led them to conclude that the disease caused by F. oxysporum

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63 Candidate Biological Control Agents against Cannabis and Coca f.sp. cannabis was not severe enough to cause “permanent and lasting control” of cannabis plants and that there was a need for an improved formulation and improved delivery systems to enhance the pathogen’s efficacy as a mycoherbi- cide (Tiourebaev et al. 2001). The results of those studies are reviewed below in the context of efficacy, inoculum production and delivery, and persistence in the environment. No studies of F. oxysporum f.sp. cannabis for the control of can- nabis have been published since 2001. Biological Control of Coca with Fusarium oxysporum f.sp. erythroxyli Sands et al. (1997) isolated F. oxysporum from severely diseased coca plants (Erythroxylum coca and E. novogranatense) grown by the U.S. Depart- ment of Agriculture (USDA) on a secure experimental site on the island of Kauai, Hawaii. This site was originally a coca plantation of a U.S. beverage (soda) manufacturer in the 1960s and is now maintained by USDA for its herbi- cide studies on coca. Coca plants grown on the site (grown from seeds imported mainly from Peru) in the 1960s and 1970s exhibited damping off and wilt symp- toms (Darlington 1996). Pathogenicity and host-range experiments led the inves- tigators to identify the F. oxysporum as a unique forma specialis that attacks members of the family Erythroxylaceae and to name it F. oxysporum f.sp. erythroxyli (Sands et al. 1997). In Peru, a similar disease of coca plants was observed in the 1930s (El Comercio 1995), in the 1980s (Stevenson 1991), and in the 1990s (Arévalo et al. 1994). F. oxysporum was the causal agent of an epidemic in coca in the Hual- laga Valley of Peru, and the disease was considered a threat to coca production in that region (OTA 1993; Arévalo et al. 1994). In 1997, with the aid of random amplified polymorphic DNA (RAPD) analysis, Nelson et al. identified two sub- populations of F. oxysporum f.sp. erythroxyli in the Huallaga Valley (Nelson et al. 1997). Later, Gracia-Garza et al. (1999) used RAPD and vegetative compati- bility group (VCG) analyses to determine that F. oxysporum f.sp. erythroxyli isolates from Peru and Hawaii were genetically similar. They speculated that the pathogen might have been introduced into Hawaii in plant material from Peru (Gracia-Garza et al. 1999). The known distribution of F. oxysporum f.sp. erythroxyli today includes Peru, Hawaii, and possibly Colombia (El Comercio 1995; Nelson et al. 1997). On the basis of its ability to cause the observed natu- ral wilt disease in coca plantings in Hawaii and Peru and its purported narrow host range, it is considered a potential mycoherbicide for coca (Bailey et al. 1997; Sands et al. 1997). Several studies of F. oxysporum f.sp. erythroxyli were conducted in the United States in the middle to late 1990s to develop methods for inoculum pro- duction and formulation for the purpose of evaluating its efficacy against coca (Bailey et al. 1997; Hebbar et al. 1997; Connick et al. 1998) and to study its dis- persal in the field (Bailey et al. 1997; Bailey et al. 1998; Gracia-Garza et al. 1998, 1999). The results of the studies are reviewed below in the context of effi-

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64 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops cacy, inoculum production and delivery, and persistence in the environment. No studies of F. oxysporum f.sp. erythroxyli for the control of coca have been pub- lished since 1999. EFFICACY AND IMPLEMENTATION The overarching consideration in determining the feasibility of the pro- posed mycoherbicides is the ability of F. oxysporum f.sp. cannabis and F. ox- ysporum f.sp. erythroxyli to inflict severe damage or death on their target plants. The committee reviewed the available data for evidence of such high levels of efficacy as a prerequisite for the use of the identified strains as mycoherbicides. In the case of registered mycoherbicides—such as Chontrol, Collego (Lock- down), DeVine, and Sarritor—the most commonly used measure of efficacy is reduction in weed numbers due to complete and fairly rapid killing of the target weed after application. Accordingly, in the following analysis, efficacy is viewed as the ability to yield an acceptable level of control of the target crops within a short period after application, namely, a few days to a few weeks for the annual crops of cannabis and a few months in the case of coca, a long-lived tree crop. As mentioned in Chapter 1, eradication of the crops is not a realistic goal, because biological agents rarely, if ever, kill 100% of their hosts. There- fore, the committee looked for data on different steps in efficacy measurements, such as greenhouse and field evaluations, with emphasis on the latter; appraisal at relevant growth stages of the target crops; and suitable measures of control, such as plant death and reduction in plant population density, plant growth rate, or crop yield (as harvestable biomass of cannabis and coca). Disease assess- ments reported in the literature, including visual ratings and direct measurement of disease on individual plants and populations, help to quantify the destructive- ness of the pathogens in controlled experiments, but they provide only an indi- rect measure of the potential efficacy of the proposed mycoherbicides under field conditions. Fusarium oxysporum f.sp. cannabis Only two publications shed light on the efficacy of F. oxysporum f.sp. cannabis as a mycoherbicide for cannabis. McCain and Noviello (1985) in con- ference proceedings reported on the effectiveness of F. oxysporum f.sp. canna- bis against cannabis (industrial hemp) plants in the greenhouse, growth chamber, and fields in Italy. Tiourebaev et al. (2001) in a short communication described greenhouse and field studies to evaluate the pathogenicity and virulence of F. oxysporum f.sp. cannabis isolates obtained from diseased cannabis plants col- lected in various regions of Kazakhstan. The details of the experimental methods used in those studies are presented in Tables 4-1 and 4-2.

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65 Candidate Biological Control Agents against Cannabis and Coca TABLE 4-1 Greenhouse and Field Studies in Italy by McCain and Noviello (1985) Greenhouse Study Inoculum F. oxysporum f.sp. cannabis isolate recovered from industrial hemp plants collected from fields in Italy in 1972 Inoculum (composed of mycelium, conidia, and chlamydospores) produced on wheat straw and soybean meal Five inoculum levels tested: 7, 70, 700, 1,400, and 7,000 propagules/g of soil Test plants C. sativa cultivar Iran (susceptible variety) Assessment method Efficacy based on percentage of plants killed Field Studies Location Field experiments conducted in Vitulazio, Alvignano, and Portici, Italy, in 1974 Inoculum Air-dried straw-soybean inoculum used; applied by hand and mixed with top 10 cm of soil Vitulazio field trial: 10 g/m2; inoculated plots seeded with C. Application rate and cultivars used sativa cultivars CS and SF Alvignano field trial: 10 g/m2; inoculated plots seeded with C. sativa cultivars Iran and SF Portici field trial: 1, 10, and 30 g/m2; inoculated plots seeded with C. sativa cultivars Iran, CS, and SF; trial repeated with field soil in large ceramic pots Assessment method Efficacy based on plant mortality In their greenhouse study, McCain and Noviello noted that the time re- quired to kill all the seedlings was proportional to the inoculum level. At the highest inoculum level (7,000 propagules/g of soil), 100% of the plants were killed within 9 days after planting, whereas it took 47 days for all the plants to die at the inoculum level of 70 propagules/g of soil. Only 50% of the plants were killed at the lowest inoculum level (7 propagules/g of soil) at 47 days after plant- ing. McCain and Noviello reported that in field plots at Vitulazio, no Fusa- rium-infected CS or SF hemp plants were observed during the study, but they did not provide an explanation for the apparent failure of this trial. At the Alvignano field trials, 4 months after the cultivars were planted in the fungus- infested soil, 71% of the Iran plants had died and the ones that survived were shorter than the plants in the noninoculated control plots. Only two SF plants became infected—an indication that this cultivar is resistant to the disease. In the Portici trial, 50%, 94%, and 94% of the Iran plants had died 4 months after the cultivars were planted in the soil treated at 1, 10, and 30 g/m2, respectively. The Iran plants that survived were stunted by 26 and 45% at the 1-g/m2 and 10- g/m2 inoculum levels, respectively, compared with the Iran plants in the non- inoculated control plots. At the highest inoculum level, 30 g/m2, only 4% and 14% of the cultivars CS and SF died.

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66 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops TABLE 4-2 Greenhouse and Field Studies in Kazakhstan by Tiourebaev et al. (2001) Greenhouse Study Inoculum 125 isolates of F. oxysporum obtained from diseased C. sativa plants collected in various regions of Kazakhstan 125 isolates of F. oxysporum “evaluated for pathogenicity” to C. sativa All isolates formulated with wheat or oat seeds or birch sawdust and applied at 0.5 g per 2-cm2 pot of soil Test plants C. sativa (cultivar not stated) Environmental conditions 28°C; 19-h photoperiod Assessment method Disease severity rating: 1 = healthy plant; 2 = wilting of lower leaves; 3 = wilting of 25-50% of leaves; 4 = over 50% of leaves wilted; 5 = dead plant Field Studies Location Kazakhstan field naturally infested with C. sativa plants (cultivar not stated) Inoculum 25 F. oxysporum isolates “that showed virulence and host specificity towards C. sativa” in the greenhouse Trial 1: 25 isolates formulated with wheat seed, oat seed, and birch sawdust Trial 2: 25 isolates formulated in birch sawdust 12.5 g/m2 (plot size, 2 m2); inocula of the 25 isolates applied twice: Application rate when C. sativa seedlings were 2-3 wk old and when the plants were 6-7 wk old Assessment method Estimation of “percentage of infected plants within treated plots” done on 2-m transect laid across plot; disease severity calculated with the equation D = a/b(100), where D = disease severity, a = number of C. sativa plants with disease severity rating of 3-5 (see rating scale in greenhouse studies), and b = total number of C. sativa along the transect with 1-5 disease severity rating There are a few important shortcomings in the experiments and paper by McCain and Noviello (1985): the greenhouse trial of the inoculum rate study was done only once, and the number of replications per treatment was not stated; the temperature and humidity conditions during the greenhouse and field trials were not stated; and the isolates used were not specifically identified. Thus, the experiments are not readily repeatable, and the results cannot be accepted as critical evidence. McCain and Noviello screened several other unnamed C. sativa cultivars besides those in Table 4-1 and seed collections and discovered that some indus- trial hemp varieties were resistant to F. oxysporum f.sp. cannabis. That led them to conclude that the presence of resistant varieties might limit the effectiveness of the mycoherbicide.

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67 Candidate Biological Control Agents against Cannabis and Coca The results of the study by McCain and Noviello confirm that F. ox- ysporum f.sp. cannabis is a pathogen of C. sativa, but the pathogen’s efficacy as a mycoherbicide agent is low. By the authors’ own admission, the time required for disease development and death of cannabis (at least 4 months) is a limitation (McCain and Noviello 1985). The decline of populations of F. oxysporum f.sp. cannabis in the soil in the absence of the host plant suggests that the fungus may not persist unless the fields are continuously cropped with cannabis. The au- thors’ claim that complete coverage of a field is unnecessary because natural spread would occur is not backed up by the data that they presented. Therefore, the hypothesis that F. oxysporum f.sp. cannabis will persist in treated soil and can infect new crops of cannabis remains untested. Tiourebaev et al. (2001) conducted greenhouse and field experiments with a fairly large collection of F. oxysporum f.sp. cannabis isolates. In the green- house study that was conducted only once, 25 of the 125 isolates tested were found to cause disease in cannabis. Plant mortality for the wheat-seed, oat-seed, and birch-sawdust formulations was 64%, 61%, and 59%, respectively. Tioure- baev et al. noted that cannabis mortality varied according to the fungal isolate. In the first field experiment, 12 of the 25 isolates that were previously screened in the greenhouse study were found to be pathogenic to cannabis. Iso- late CR-21 caused the most damage, namely, wilting of the plant within 2 weeks. Other symptoms on the treated plants were stunting, leaf curling, root discoloration, and loss of structural integrity in the upper part of the plant. At the end of the first field experiment (5 months after the initial application of the formulations), disease incidence ranged from 12% to 67% for the isolates tested. Disease severity ranged from 1.27 to 2.00 on a scale of 1-5 (see Table 4-2), in which a rating of 2.00 corresponds to wilting of lower leaves. In the second field trial, disease incidence ranged from 6.8% to 39% and disease severity from 1.4 to 2.7 for the isolates tested. Plot size was 2 m2, and the number of replicates was not mentioned (Tiourebaev et al. 2001). Like McCain and Noviello, Tiourebaev et al. (2001) confirmed F. ox- ysporum f.sp. cannabis as a pathogen of cannabis. However, the levels of dis- ease, based on disease-severity and disease-incidence estimates, were low to moderate and led the authors to conclude that under field conditions “the infec- tion rate was still too low to affect permanent and lasting control of the weed.” Thus, the Kazakh isolates of F. oxysporum f.sp. cannabis tested by Tiourebaev et al. (2001) do not appear to be efficacious or suitable for development as a mycoherbicide. According to Tiourebaev et al. (2001), the “pathogenicity of the isolates varied greatly” between greenhouse and field trials. One explanation for the variation could be temperature: the greenhouse temperature during the experi- ment was 28°C with 19 hours of daylight, whereas the temperature in the field ranged from 20°C to 30°C during May or June, the time when infection began to appear. The disease levels were highest in late August and September and, ac- cording to the authors, the “disease tapered off as cooler conditions prevailed”

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68 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops (Tiourebaev et al. 2001). Another factor might be the genetic composition of the cannabis plants in the field; inasmuch as the trial took place in a field with a natural cannabis infestation, the hosts’ genetic variability could have affected the isolates’ field performance. The identification of the isolates as F. oxysporum was based on “micro- scopic analysis and colony morphology” (Tiourebaev et al. 2001). The authors assigned the name F. oxysporum f.sp. cannabis without completing Koch’s pos- tulates to prove their pathogenicity toward cannabis. Without additional support from molecular or VCG analysis, this identification is unlikely to be acceptable for mycoherbicide-registration purposes. It cannot be ruled out that this large collection of F. oxysporum isolates from the wild included several formae spe- ciales that are cross-infective on cannabis and one or more additional hosts, with cannabis being an alternative rather than a primary host. Overall, the data on F. oxysporum f.sp. cannabis gleaned from the publi- cations of McCain and Noviello (1985) and Tiourebaev et al. (2001) are not sufficient to draw conclusions about the feasibility of F. oxysporum f.sp. canna- bis as a mycoherbicide. Fusarium oxysporum f.sp. erythroxyli The committee reviewed the available data on the efficacy of F. ox- ysporum f.sp. erythroxyli in three publications: Sands et al. (1997), Bailey et al. (1997), and Bailey et al. (1998). The study by Sands et al. consisted of experi- ments in the field and in a growth chamber to assess the virulence of F. ox- ysporum f.sp. erythroxyli isolated from an Erythroxylum population growing in Hawaii. Bailey et al. (1997) performed three field trials in Hawaii with a rice- alginate prill formulation of strain EN-4 of F. oxysporum f.sp. erythroxyli, which was one of the Hawaiian isolates studied by Sands et al. (1997), to test the feasibility of enhancing the pathogen populations in the soil and to cause disease in coca. Later, Bailey et al. (1998) examined six formulations, including the rice-alginate prill, for their ability to enhance the pathogen’s populations and cause disease on coca in the field. The experimental methods used in the studies are summarized in Tables 4-3 and 4-4. In the growth-chamber study by Sands et al. (1997), severe disease was not observed with any of the F. oxysporum f.sp. erythroxyli strains tested when the inoculum was placed in 10-cm holes around the plant. Better results were obtained by first infesting the soil with the fungus and then transplanting coca plants into the soil. Wilting was observed 3 weeks after the plants were trans- planted, and the plants eventually died; however, no quantitative data were pro- vided. In the field study, the initial disease symptoms were leaf drop and death of a “few lower stems”; plant death was observed 7 weeks after the plants were transplanted into the fungus-infested soil. The time from the appearance

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69 Candidate Biological Control Agents against Cannabis and Coca TABLE 4-3 Growth-Chamber and Field Studies by Sands et al. (1997) Growth-Chamber Study Inoculum F. oxysporum strains tested: Ec1-3 and EN1-4 (from Hawaii) and SA1 (from South America) Soil in pots planted with test plants infested with millet seeds colonized by F. oxysporum by placing seeds in three 10-cm holes (10 seeds/hole) Test plants 6- to 12-mo E. coca plants Environmental conditions 30°C/28°C day/night temperature; 12-h light/dark periods Test plants watered daily (80-100 mL) Assessment method Test plants evaluated for disease weekly over 3.5-mo period Field Study Location Conducted in 1989 on field plots transplanted with E. coca and E. novogranatense in the previous fall (1988) Inoculum Field plots infested with millet seeds colonized by F. oxysporum Seven Hawaiian isolates of F. oxysporum evaluated for pathogenicity on coca Environmental conditions All infested plots received trickle irrigation Application method Subsurface method: inoculum (5.0 g /plot) deposited in and rate 2-cm-deep V-shaped trench and covered with 2-3 cm of soil Surface method: inoculum applied directly to the soil surface and covered with mesh screen to protect from predation Assessment method Plants in treated and nontreated soil evaluated for severity and mortality after 7, 8, 9, 10, and 15 mo Efficacy assessed with a disease-severity rating scale of 0-2: 0 = no disease, 1 = wilt, 2 = plant death of initial disease symptoms to plant death ranged from days to months. The av- erage disease severity for all seven isolates tested was 1.1 (on a scale of 0-2) in the subsurface-applied plots and 1.03 in the surface-applied plots. Plants in the control plots had an average disease severity rating of 0.71, that is, plants were symptomatic but not dead. Some control plants also developed disease, pre- sumably from naturally occurring inoculum of F. oxysporum f.sp. erythroxyli. E. coca plants were more severely diseased (rating, 1.22) than E. novogranatense plants (0.93). Apparently on the basis of a combination of the data from all seven iso- lates and two methods of inoculum application, Sands et al. (1997) reported that 94% of E. coca and 49% of E. novogranatense plants (42 per species) in the fungus-infested soil were killed at 15 months after inoculation, whereas about 95% of E. coca and 43% of E. novogranatense plants (28 plants per species) in the noninfested control soil (values read off the graph in Figure 1 of Sands et al. [1997]) had died. F. oxysporum was isolated from the vascular tissues of symp- tomatic and asymptomatic plants from the fungus-infested soil and from symp-

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70 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops tomatic plants in the noninfested soil; this implied nonpathogenic colonization of coca plants by F. oxysporum f.sp. erythroxyli and fairly high levels of natural incidence of wilt at the test site. TABLE 4-4 Field Studies in Hawaii by Bailey et al. (1997, 1998) First Set of Field Trials Location Three experiments conducted from 1995 to 1996 in Kauai, Hawaii Two trials in fields continuously planted with coca for at least 7 years; one trial in a field not previously planted to coca Environmental conditions Fields irrigated daily for 14 d after F. oxysporum f.sp. erythroxyli strain EN-4 formulations were incorporated in soil Temperature 17-21°C minimum and 23-27°C maximum; regular afternoon rains (3.9-57.9 cm) Inoculum Alginate prill formulation F. oxysporum f.sp. erythroxyli strain EN-4 Application rate 33.6 kg/ha Assessment method F. oxysporum f.sp. erythroxyli strain EN-4 efficacy assessed with disease-severity rating scale of 0-2: 0 = asymptomatic, 1 = symptomatic (plant defoliating), 2 = dead Second Set of Field Trials Location Three experiments in 1995-1996 in Kauai, Hawaii Two experiments in fields continuously planted with coca for at least 7 years; one trial in a field not previously planted to coca Low percentage of diseased coca plants present in all field plots before the experiment was conducted Environmental conditions Fields irrigated daily for 14 d after the F. oxysporum f.sp. erythroxyli strain EN-4 formulations were incorporated in soil For experiments 2 and 3, average low air temperature: 19.5°C and 21.3°C; average low soil temperature: 22.6°C and 24.2°C; relative humidity for both trials from less than 80% to over 97% during the first 10 d after application of formulations Inoculum Six formulations (rice-alginate, C6, Pesta, canola-alginate, rice-alginate + canola oil, corn cob-alginate) and biomass alone of F. oxysporum f.sp. erythroxyli strain EN-4 Application rate 33.6 kg/ha Assessment method F. oxysporum f.sp. erythroxyli strain EN-4 efficacy assessed with disease-severity rating scale of 0-2: 0 = asymptomatic, 1 = symptomatic (plant defoliating), 2 = dead

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71 Candidate Biological Control Agents against Cannabis and Coca These results confirm F. oxysporum f.sp. erythroxyli as a virulent patho- gen of E. coca and E. novogranatense; with respect to efficacy, however, the data are equivocal. No quantitative data from the growth-chamber study were provided, so these data are of limited value in assessing efficacy. The data in Figure 1 of the paper shows that the percentages of dead control and fungal- treated E. coca plants were nearly equal at the end of the experiment. In the case of E. novogranatense, nearly 60% (not 49% as stated in the publication) of the inoculated plants and nearly 50% of the control plants appear to have died. Without the benefit of statistical analysis of data in Figure 1, the small number of plants tested (42 and 28 plants per species in the fungus-infested and fungus- free soils, respectively) raises the question whether the naturally prevalent in- oculum at the field site rather than the experimentally applied inoculum was responsible for the observed results. Furthermore, the lack of information on climatic conditions (average daily temperature, relative humidity, soil moisture) during the study and the use of 6- to 12-month-old seedling transplants instead of older and established coca plants are additional limitations. In two later studies, Bailey et al. (1997, 1998) reported an increase in the rate of disease development in plots treated with different formulations com- pared with the rate in untreated plots, which had only background levels of Fusarium wilt. However, the mortality of coca plants was variable, ranging from 35% to 85% among all seven formulations tested (Bailey et al. 1998). The results obtained by Bailey et al. are consistent with the finding by Sands et al. (1997) that F. oxysporum f.sp. erythroxyli is a virulent pathogen of coca plants. However, as in the study by Sands et al., the results with respect to efficacy are ambivalent. Bailey et al. (1997) claimed that “the 33.6 kg/ha rate of the rice-alginate prill formulation enhanced the killing of coca plants in three different field experiments over 7-9 months,” but the data in the paper do not report plant mortality; the maximum disease rating for up to 350 days was under 1; that is, the plants were symptomatic, not dead. Bailey et al. (1997) also re- ported that some plants “appear to seal off the infected areas and resume normal growth patterns,” and this could indicate the plants’ ability to overcome the dis- ease. The incidence of natural wilt of about 58% at the experimental site in Ha- waii where Bailey et al. (1997) performed the first set of field studies and the 17% mortality in the second set of field studies (Bailey et al. 1998) are problem- atic. Against such background disease levels, the increase in disease incidence resulting from the added inoculum cannot be ascertained on the basis of the col- lective data of Bailey et al. (1997, 1998) and Sands et al. (1997). Bailey et al. (1997) also state that by “maintaining soil surface moisture with drip irrigation, overhead watering, and/or rain, the rice-alginate prill formu- lation germinated relatively uniformly in two greenhouse experiments and at least two field experiments when applied to the soil surface.” Thus, the rice- alginate prill formulation appears to be a feasible and effective way to deliver the inoculum if it is deployed under conditions where adequate moisture is pre- sent.

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90 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops Transmission and Spread The potential pathways by which spores and other propagules from a par- ticular application site might move via physical transport mechanisms are illus- trated in Chapter 2 (see Figure 2-1). In general, dispersal of the proposed myco- herbicides after application will depend on the production and natural dispersal of secondary inoculum. In the case of F. oxysporum, secondary spread was originally thought to be limited to root-to-root contact and distribution of in- fected plant tissues and seeds (Gambogi 1983; Gracia-Garza et al. 1999; Rekah et al. 2001). However, additional evidence indicates that under humid condi- tions, several formae speciales of F. oxysporum produce conidia in stroma (mass of hyphae) on the lower stems of infected plants (Rowe et al. 1977; Timmer 1982; Woudt et al. 1995; Gamliel et al. 1996; Vakalounakis 1996; Katan et al. 1997; Rekah et al. 1999, 2000, 2001; El-Hamalawi and Stanghellini 2005). These conidia can be dispersed by rain, wind, or insects (Gillespie and Menzies 1993; El-Hamalawi 2008). Inoculum could also be transferred from the applica- tion site by several vectors, including humans (transporting plant material or infested seeds), the hides or fur of mammals (such as pack animals), the drop- pings of birds, or the surface of insects. Long-distance transport via waterways is a potential route inasmuch as viable and infectious propagules of F. ox- ysporum have been found in rivers and seas (Palmero et al. 2009), although the distance over which such transport might occur is unknown. Finally, windstorms could carry inoculum over long distances to areas far outside the target areas (Maldonado-Ramirez et al. 2005). Thus, the potential exists for the proposed mycoherbicides to move out of the geographic areas where they are applied. Only a few studies have examined the dispersal of F. oxysporum f.sp. cannabis and F. oxysporum f.sp. erythroxyli. In a field study in Kazakhstan, consumption and dispersal by insects, lizards, rodents, and birds was reported for a seed-based inoculum of F. oxysporum f.sp. cannabis (Tiourebaev et al. 2001). In a field study in Hawaii, the applied inoculum of F. oxysporum f.sp. erythroxyli was foraged by ants and became clustered in their nests (Gracia- Garza et al. 1998). In addition, many Fusarium species, including F. oxysporum, survive on and in seeds (Nelson et al. 1997; Garcia-Garza et al. 1999; Mbofung and Pryor 2007). Transmission by seeds and planting materials carries the risk of unin- tended spread beyond target areas in that farmers and traders could carry in- fected materials throughout a region or even into new areas, as has been pro- posed for the transport of F. oxysporum f.sp. erythroxyli that appears to have arrived in Hawaii from Peru (Fravel et al. 1996; Sands et al. 1997). Other Host Plants In general, Fusarium species are ubiquitous in the environment and can survive on host plants that may or may not be susceptible to them (Blok and

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91 Candidate Biological Control Agents against Cannabis and Coca Bollen 1997). In a host-range study of F. oxysporum f.sp. erythroxyli, Sands et al. (1997) reported that the pathogen was isolated from the crown tissue of symptomless plants of 26 species grown in treated soil. Thus, this fungus could colonize plants other than coca. However, no studies have evaluated the long- term persistence of either F. oxysporum f.sp. erythroxyli or F. oxysporum f.sp. cannabis through observations on their survival in soil after the host plant dies or evaluated their potential to colonize later plantings of coca or cannabis. The recent study of genetic variation at two loci among F. oxysporum in- dividuals pathogenic on animals and plants (O’Donnell et al. 2009) showed that F. oxysporum f.sp. erythroxyli has a unique genotype at both loci but that F. oxysporum f.sp. cannabis shared one or both alleles with individuals that were isolated from other plants. Although it certainly is possible that either Fusarium mycoherbicide could attack nonhost plants, it also is true that the mycoherbicide strains would not be expected to behave differently from members of the two Fusarium species that are already present in the endemic areas. Competition with Other Soil Microorganisms Although it was demonstrated that F. oxyspsorum f.sp. erythroxyli can be suppressed in some soil types (Fravel et al. 1996), no studies are available on the interactions of F. oxysporum f.sp. cannabis or F. oxysporum f.sp. erythroxyli with particular soil microorganisms or other organisms. Studies of other F. ox- ysporum formae speciales and other fungi suggest that the presence of competi- tor or antagonistic microorganisms could reduce persistence. For example, springtails and mites feed on plant pathogenic fungi (Nakamura et al. 1992; Okabe 1993), and several bacteria and fungi can suppress the pathogens respon- sible for Fusarium wilt of melon (Suárez-Estrella et al. 2007), tomato (Larkin and Fravel 1999), and chickpea (Landa et al. 2004). In fact, such antagonists have been pursued as biological control agents against these wilt diseases. Thus, antagonistic microorganisms in the soil could theoretically lessen the likelihood that the proposed mycoherbicides would establish sufficiently high inoculum levels in the rhizosphere to cause wilt disease of cannabis or coca. In contrast, it is possible that the mycoherbicide strains could displace resident strains; but, again, no data are available to evaluate this possibility. Conclusions It is clear that F. oxysporum f.sp. erythroxyli has the ability to colonize and survive in soil and coca roots for several months, but there are insufficient data to predict the long-term persistence of F. oxysporum f.sp. erythroxyli, in that none of the studies lasted for more than 7 months. However, the natural presence of this fungus and the recurrent incidence of the coca wilt disease in the Huallaga Valley of Peru (see discussion later in this chapter) suggest that F. oxysporum f.sp. erythroxyli is capable of long-term persistence. Inasmuch as F.

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92 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops oxysporum f.sp. erythroxyli could survive on plants other than coca and on de- caying organic material, it is likely that the mycoherbicide strain would persist in the indigenous fungal populations at some level once it is introduced in coca fields. In general, the environmental factors that affect the persistence of F. ox- ysporum f.sp. erythroxyli may also affect F. oxysporum f.sp. cannabis, but too little information is available on the latter forma specialis to draw any conclu- sions beyond this generalization. EFFECTS ON NONTARGET ORGANISMS Microbial pesticides are regulated by the U.S. Environmental Protection Agency, which requires a variety of testing of 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, toxicity and pathogenicity testing on nontarget organisms, and assessment of environmental fate. The aforementioned studies have not been conducted with the goal of registering the tested strains of F. oxysporum f.sp. cannabis or F. oxysporum f.sp. erythroxyli as mycoherbicides, so this section reviews the existing data on them that are available in the open literature and are pertinent to understanding potential adverse effects on nontarget plants and or- ganisms. Consideration is given to the issues specified in the committee’s state- ment of task, including potential effects on licit crops, other soil fungi, animals, humans, biodiversity, and other relevant aspects of environmental health. Effects on Nontarget Plants Some research has been performed to evaluate the effects of F. oxysporum f.sp. cannabis and F. oxysporum f.sp. erythroxyli on native plant species, but the reported data are sparse. For example, Tiourebaev et al. (2000, 2001) tested F. oxysporum f.sp. cannabis on several plant species in Kazakhstan, including food crops and native plants (see Table 4-11). The first paper appeared in the pro- ceedings of a conference and reported that none of 13 plants tested exhibited symptoms of Fusarium wilt but provided no experimental data to support the claim (Tiourebaev et al. 2000). The second paper reported that host-range tests were conducted under greenhouse conditions on five crops that were commonly cultivated in the test region of Kazakhstan (tomato, corn, wheat, bean, and po- tato) but contained no data or discussion of the results obtained pertaining to those crops (Tiourebaev et al. 2001); instead, the authors simply state that 12 nontarget plants “showed no evidence of infection by the pathogen” and seemed to imply that the results were the results of field tests. The possible taxonomic and etiological relationships of F. oxysporum f.sp. cannabis to other wilt-causing Fusarium species that attack plants related to cannabis, such as the economically important hops (Humulus lupulus), were not

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93 Candidate Biological Control Agents against Cannabis and Coca adequately addressed either by McCain and Noviello (1985) or by Tiourebaev et al. (2001). The claim by McCain and Noviello that hops “are not known to be susceptible to a [Fusarium] wilt disease anywhere in the world” is erroneous (McPartland and West 1999; Solarska 2001). One host-range study of F. oxysporum f.sp. erythroxyli was identified. Sands et al. (1997) tested strains of this fungus from Hawaii and South America on 26 plant species (see Table 4-12) grown in growth chambers from seeds planted in pathogen-infested soil. Wilt symptoms were not observed on any of the native species. However, when crown tissues of the species grown in the infested soil were plated on a selective medium at the conclusion of the experi- ment, F. oxysporum was isolated from all species. Those results indicate that F. oxysporum f.sp. erythroxyli was not pathogenic to the tested hosts, but because it was present in the crowns the authors concluded that it could infect nontarget host tissues. TABLE 4-11 Plants Reportedly Tested in Host-Range Studies of Fusarium oxysporum f.sp. cannabis Tiourebaev et al. 2000 Tiourebaev et al. 2001 Agropyron spicatum (wheat grass) Crops: Cucumus sativus (cucumber) Lycopersicon esculentum (tomato) Daucus carota (wild carrot) Phaseolus vulgaris (kidney bean) Festuca arundinaceae (tall fescue; grass) Solanum tuberosum (potato) Gossypium hirsutum (cotton) Triticum aestivum (wheat) Hordeum vulgare (barley) Zea mays (corn) Lycopersicon esculentum (tomato) Native plants: Melilotus indica (sweet clover; herb) Agropyrum pectiniforme (crested wheatgrass) Phaseolus vulgaris (kidney bean) Alhagi pseudalhagi (camelthorn; legume) Pisum sativum (garden pea) Artemisia vulgaris (common wormwood; Raphanus sativus (radish) herbaceous perennial) Triticum aestivum (wheat) Atriplex alba (lambsquarters goosefoot; shrub) Zea mays (corn) Bromus inermis (awnless brome; grass) Ceratocarpus arenarius (jiao gulo li; herb) Chenopodium sp. (common lamb’s quarters; herbaceous annual) Dactylis glomerata (orchardgrass) Glycyrrhiza glabra (cultivated licorice; legume) Kochia prostrata (forage kochia; shrub) Stipa dasyphylla (grass) Tragopogon major (yellow salsify)

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94 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops TABLE 4-12 Plants Reportedly Tested in Host-Range Studies of Fusarium oxysporum f.sp. erythroxyli Abelmoschus esculentus (okra) Helianthus annuus (sunflower) Allium cepa (onion) Hordeum vulgare (barley) Arachis hypogaea (peanut) Lactuca sativa (lettuce) Beta vulgaris (sugar beet) Lycopersici esculentum (tomato) Capsicum annuum (cayenne pepper) Oryza sativa (rice) Carthamus tinctorius (safflower) Phaseolus vulgaris (kidney bean) Citrullus lanatus (watermelon) Pisum sativum (garden pea) Cucumis melo var. cantalupensis (cantaloupe) Raphanus sativus (radish) Cucumis sativus (cucumber) Sorghum bicolor (sorghum) Cucurbita moschata (pumpkin) Triticum aestivum (wheat) Daucus carota subsp. sativus (carrot) Vigna unguiculata (cowpea) Glycine max (soybean) Zea mays (corn) Gossypium barbadense (annual long-fiber cotton) Source: Adapted from Sands et al. 1997. Overall, the data on the host range of F. oxysporum f.sp. cannabis and F. oxysporum f.sp. erythroxyli are insufficient to draw any conclusions. Erythroxy- lum is a large genus; it has as many as 250 species, of which about 200 have the same native habitat as the coca plants (Plowman 1980). Thus, it is important to determine whether F. oxysporum f.sp. erythroxyli could attack potential alterna- tive hosts and whether any of them are of ecological significance, for example, endangered, threatened, or of particular value. Conducting such tests and interpreting the data from them may be difficult because F. oxysporum is ubiquitous and “the host range tests required to prove specificity [of the formae speciales] would be very large and likely prohibitive” (Berner and Bruckart 2005, p. 227). Effects on Legal Crop Production The potential risk to legal production of cannabis and coca has not been given much attention. Commercial cultivation of hemp is not legal in the United States, but hemp is grown in Canada and many other countries (for example, several European countries, China, and Russia) for fiber, for linen production, and for seeds, which are a source of meal and oil used in food and other prod- ucts (USDA 2000). McCain and Noviello (1985) claimed that “because of the restricted host range, no special precautions are necessary for controlled use of the fungus,” even though the industrial hemp cultivars tested developed 10-100% disease. They also claimed that cannabis cultivars or seed collections from Czechoslova- kia, India, Iran, Mexico, Nepal, Pakistan, Poland, South Africa, Thailand, and

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95 Candidate Biological Control Agents against Cannabis and Coca Turkey were “all highly susceptible to the disease and in general there were no survivors or escapes in [a] growth chamber study.” However, no data were pro- vided on the extent of cultivar differences in susceptibility. On the basis of the McCain and Noviello (1985) data, F. oxysporum f.sp. cannabis could pose a risk to commercial hemp grown for fiber. Toxicity to Wildlife, Domestic Animals, and Humans This subsection focuses on the potential toxicity to nontarget organisms. As discussed in Chapter 1, toxicity refers to the degree or extent of harm caused by a chemical. In the case of the proposed mycoherbicides, the greatest risk to animals and humans probably would come from the consumption of the patho- gen in colonized plant material. Toxins might be produced in the materials used to deliver the inoculum. Tiourebaev et al. (2001) found that when F. oxysporum f.sp. cannabis was applied to wheat and oat seeds, insects, lizards, and rodents consumed the inoculum. There are no studies of mycotoxin production by F. oxysporum f.sp. cannabis or F. oxysporum f.sp. erythroxyli. As noted earlier in this chapter, F. oxysporum is a large and diverse species complex, and the genus Fusarium is even larger, so generalizations or assumptions about the production of mycotoxins by the proposed mycoherbicide strains cannot be made, even though there is a great deal of information about mycotoxins produced by mem- bers of the genus Fusarium (Marasas et al. 1984; Desjardins 2006). Filamentous fungi produce a variety of metabolites. Primary metabolites are chemicals that are required for the survival of an organism, and secondary metabolites are organic compounds whose production by an organism is not required for growth, development, reproduction, or immediate survival. Biosyn- thesis of secondary metabolites often begins with components derived from pri- mary metabolism. Nearly all fungi produce at least one secondary metabolite. Although the functions of most secondary metabolites are unknown, many have been shown to be biologically active. Many have antimicrobial activity and thus might play a role in competition with other microorganisms. Others are toxic to plants and function as pathogenicity factors. Secondary metabolites that are toxic to animals and humans are called mycotoxins. Mycotoxins are grouped into classes on the basis of their chemical structure. All members of a class have a common core structure; each member has its own unique chemical modifications of the common core. The toxicity of members of a class is diverse; they can range from nontoxic to lethal. Sensitivity to mycotoxins also varies: some animals exhibit acute reactions to a particular mycotoxin and members of other species find it innocuous or require long expo- sures to produce any observable symptoms. Some members of the genus Fusarium produce toxic secondary metabo- lites (Marasas et al. 1984; Desjardins 2006). Table 4-13 presents a summary of the classes of mycotoxins reportedly produced by F. oxysporum. Most research

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96 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops TABLE 4-13 Classes of Mycotoxins Produced by Fusarium oxysporum Mycotoxin Class Source Reference Beauvericin Maize culture Logrieco et al. 1998 Fumonisins Wheat culture Seo et al. 1996 Fusaric acid Maize culture Bacon et al. 1996 Fusarin C Defined medium Cantalejo et al. 1999 Moniliformin Rice culture Abbas et al. 1990 Trichothecenes Defined medium Cantalejo et al. 1999 Wortmannin Rice culture Abbas and Mirocha 1988 on Fusarium mycotoxins has focused on the ones that contaminate important food and feed crops, such as corn, wheat, barley, and potato. Some highly toxic metabolites—such as trichothecenes, moniliformin, fumonisins, fusarin C, and zearalenone—have been found (Kim and Lee 1994; Cantalejo et al. 1999; Seo et al. 1999; Miller 2002; Rheeder et al. 2002). Trichothecene toxins include T-2 toxin and diacetoxyscirpenol, which are on the U.S. select-agents list and pose severe threats to animal or plant health or products. T-2 and diacetoxyscirpenol are most commonly produced by Fusa- rium species other than F. oxysporum. The data on trichothecene production in the report by Cantalejo et al. (1999) are questionable because the identification of the one F. oxysporum isolate to produce trichothecene was based solely on morphologic characteristics of cultures that were not grown under the standard conditions (Leslie and Summerell 2006) used for the accurate identification of these fungi. Given that background, it is unlikely that any F. oxysporum strains proposed as mycoherbicides would mutate to produce T-2 or diacetoxyscirpenol, although it would be straightforward to test mycoherbicide strains for these molecules. Because the biosynthetic pathways for these toxins are known (e.g., Desjardins et al. 1993; Kimura et al. 2007), sequencing of appropriate portions of the genomes of the proposed mycoherbicide strains could be used to deter- mine whether the genes encoding the enzymes in these pathways are present. If some of or all the necessary genes are missing, it is unlikely that the strains could mutate to produce either of the toxins. Fumonisins are a large family of related compounds, a few of which are produced by some strains of F. oxysporum. The analogues most commonly as- sociated with human health problems are those in the fumonisin B group. F. oxysporum strains are known to produce members of the fumonisin C series (Seo et al. 1996; Sewram et al. 2005), but these toxins are not produced at high concentrations even under optimal conditions. Some isolates have also been shown to produce small amounts of fumonisin B1/B2 in culture (Waskiewicz et al. 2010). Strains of F. verticillioides have been identified as responsible for the production of the fumonisin that caused neural-tube defects in newborns in the Rio Grande valley of Texas and in the Transkei region of South Africa (Ncayi-

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97 Candidate Biological Control Agents against Cannabis and Coca yana 1986; Hendricks 1999; Gelineau-van Wase et al. 2009). A strain of F. ox- ysporum used as a bioherbicide of striga was grown in culture medium known to encourage mycotoxin production (Savard et al. 1997). Small amounts of fusaric acid were detected but no other mycotoxins. Pathogenicity to Animals and Humans There are reports in the literature of about 150 cases of Fusarium infec- tions in immunocompetent people and 330 cases in immunocompromised peo- ple (references include Nelson et al. 1994; Guarro and Gene 1995; Baran 1997; Gianni et al. 1997; Romano et al. 1998, 2010; Sander et al. 1998; Musa et al. 2000; Bodey et al. 2002; Nucci and Anaissie 2002; Albisetti et al. 2004; Naiker and Odhav 2004; O’Donnell et al. 2004; Guilhermetti et al. 2007; Nucci and Anaissie 2007; Nucci et al. 2010). Among the cases reported in immunocompe- tent people, virtually all were nail infections (onychomycosis) with or without accompanying localized cutaneous infection (paronychia). Cases of keratitis were excluded from this review because Fusarium solani was reported as the etiologic agent in the overwhelming majority of cases. Increased Fusarium in- oculum in the soil and in the environment could potentially increase the number of these types of cases in the population of the target areas. Although the infec- tions lead to cosmetic disfigurement of affected nails, the infections are self- limited in immunocompetent hosts. In immunocompromised hosts, fusariosis is a much more serious infec- tion. Although many of the infections in this population begin as nail and super- ficial skin infections, the organism rapidly invades blood vessels and spreads hematogenously (Nucci and Anaissie 2002, 2007). These infections are difficult to diagnose and treat and lead to poor survival rates in infected patients. Al- though increased Fusarium in the environment could lead to increased infections in immunocompromised populations, the population at risk in developing na- tions would be estimated to be somewhat smaller than in developed countries, in part because of the much lower numbers of procedures performed in developing countries that involve treatments for suppressing the immune system, such as organ transplantation (Ota 2004). However, the committee is aware that the variability of conditions that contribute to immunosuppression in members of a population and the difficulty of estimation of the prevalence of such conditions clearly hinder long-term estimation of risk. A final caveat in interpreting these studies is that in many cases the species of Fusarium was not determined and in others the accuracy of the identification, which was almost always based only on structure, could be called into question. In addition, the phenotype of the poten- tial mycoherbicide strains of Fusarium appears to be largely uncharacterized. Although a few cases of infection with Fusarium have been reported in animals (for example, see Mayayo et al. 1999; Conkova et al. 2003; Evans et al. 2004; Marangon et al. 2009), most of the reports deal with cases of proven or presumed Fusarium toxicosis.

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98 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops An additional factor that should be considered in an evaluation of potential effects of Fusarium on humans is the paucity of effective antifungal drugs avail- able to treat systemic fusariosis (Azor et al. 2009). In a recent review, Lortholary et al. (2010) reported an overall rate of success of voriconazole treatment for systemic fusariosis of 47%, with a range of 0-64%, depending on the site of in- fection. Combination therapy with amphotericin B did not improve overall suc- cess. The median survival rate of patients infected with F. oxysporum was 112 days. That is perhaps not surprising, in light of the poor in vitro response of F. oxysporum isolates to antifungal drugs. Although the efficacy of voriconazole is low, it compares favorably with the administration of amphotericin B and its lipid derivatives (Pfaller et al. 2002). Voriconazole and the other mold-active azoles posaconazole and ravuconazole are likely to be the mainstays of therapy for systemic fusariosis for now (Stanzani et al. 2007). MUTATION The potential of fungi used as mycoherbicides to mutate is similar to that of other fungi in general. Fungal genetic variation can be affected by many processes, including nucleotide substitution (Kasuga et al. 2002), gene loss (Sharpton et al. 2009), gene gain by duplication (Sharpton et al. 2009), and gene gain by horizontal gene transfer (that is, not from parent to offspring) from closely related (Neafsey et al. 2010) or distantly related (Friesen et al. 2006) fungi. The gain of genetic material can be as small as a few genes or as large as complete chromosomes (Ma et al. 2010). Genetic variation can become estab- lished, or fixed, in fungal populations by natural selection or by chance. Natural selection leads to adaptation to changing environments, and the adaptation could include the ability to attack new cultivars of existing host species or new host species. Adaptation may occur in fungi that reproduce sexually or asexually, but genetic recombination speeds the process by enabling the rapid synthesis of nu- merous genotypes (Goddard et al. 2005). Recombination is accomplished by mating and meiosis but also could occur by nonsexual anastomosis and nonmei- otic recombination via the parasexual cycle (even though many fungi have mechanisms to limit nonsexual hyphal anastomosis to partners with almost iden- tical genotypes). All those evolutionary processes apply to fungi used as myco- herbicides. Strains of Fusarium have a reputation for being highly mutable in culture, especially if the strains are maintained for a long period on a medium high in simple sugars (Puhalla 1981; Leslie and Summerell 2006). Mass production of Fusarium would involve such long-term maintenance of the strains. Mutations that occur under laboratory conditions usually are of a loss-of-function nature, such as the inability to synthesize a toxin, the inability to produce one or more types of spores, abnormal hyphal structure, or loss of plant pathogenic capabili- ties. Such mutations are probably due to the inactivation of one or more func- tional genes that are already in the strain’s genome. Under field conditions, mu-

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99 Candidate Biological Control Agents against Cannabis and Coca tations would certainly occur, although there is no reason to think that they would be more likely in these fungi than in indigenous strains or any other fungi. Although no sexual stage has been reported for F. oxysporum, it is not possible to rule out its existence. For example, there are two mating types in sexual Fusarium, MAT1-1 and MAT1-2, and both are found in the strains of F. oxysporum that have been evaluated (Enya et al. 2008; Lievens et al. 2009). Therefore, it is possible that adaptation in F. oxysporum could be aided by re- combination if an as yet undetected sexual stage is occurring. It is possible that mutation could appear to be driven in F. oxysporum by selective conditions. For example, some conditions might favor a particular strain among the very large pre-existing populations of F. oxysporum in the soil. F. oxysporum is ubiquitous in soils where crops are grown, and its populations would harbor substantial genetic variation. As a consequence, the sudden ap- pearance of such a strain could be attributed to mutation even though the strain had existed at low levels for an extended period. Alternatively, phenotypic changes could result from a mutagenic mechanism, such as the one responsible for the spontaneous generation of nit mutants, which are rendered insensitive to chlorate toxicity when the strains are cultured in the presence of high concentra- tions of chlorate (Klittich and Leslie 1988). The mechanism underlying this mutagenic process has not been characterized, but it appears to have a multi- genic base (Klittich et al 1988). In both those cases, mutations appear to be di- rected by the selective conditions under which they occur. Mutations could make mycoherbicide strains more virulent and thus make a host plant more susceptible to them. It is equally likely that the host plant would produce offspring that could be more resistant to the mycoherbicide. Changes in susceptibility and pathogenicity are expected as the host-pathogen system evolves. Selection of plants resistant to mycoherbicide strains should be relatively straightforward; the more virulent the mycoherbicide strain is and the fewer genetically susceptible plants escape it by chance, the more rapidly selec- tion should occur. Mutations could affect mycotoxin production, provided that a mycoherbi- cide has the genes for toxin production. Mutations also could alter the synthe- sized toxin by blocking its biosynthetic pathway; this would lead to the accumu- lation of an intermediate in the pathway rather than of the end product. No on toxin production in the proposed F. oxysporum mycoherbicides are available. WHAT WE CAN LEARN FROM A NATURAL EPIDEMIC OF FUSARIUM OXYSPORUM F.SP. ERYTHROXYLI WILT OF COCA IN PERU The natural occurrence of diseases is common in plant populations, and plants that are intensively grown as crops are particularly vulnerable to periodic devastating epidemics. Recent history tells us that coca populations are no ex-

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100 Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops ceptions. For example, an epidemic on coca in Peru in the late 1980s was linked to F. oxysporum f.sp. erythroxyli (Arévalo et al. 2000). That natural epidemic can be viewed to some extent as a large-scale field trial that is informative in considering several questions: Did the epidemic inflict sufficient damage to re- duce crop production? Did it cause recurrent or permanent effects on coca in the affected areas? If the effects of this natural epidemic are an indication, how ef- fective would it be if the same pathogen were applied as a mycoherbicide to curtail the production of illicit-drug crops? A wilt of coca (E. coca) caused by F. oxysporum resulted in extensive losses to the coca crop in the Huallaga Valley, the main coca-producing region in Peru (Arévalo et al. 2000). Although definitive reports of the presence of the disease go back at least as far as 1932, its incidence increased sharply in the 1980s, at the time of the increase in coca production, the subsequent increase in use of agricultural chemicals, and the reduction in cultivation time (Nelson et al. 1997). The pathogen was reported as F. oxysporum f.sp. erythroxyli (Nelson et al. 1997; Arévalo et al. 2000) and one of its strains was found to be identical to a strain of the pathogen studied in Hawaii by Sands et al. (1997) and Nelson et al. (1997). The most recent epidemic of F. oxysporum f.sp. erythroxyli wilt of coca in the Huallaga Valley of Peru began in 1987. It was estimated that 52% of the coca crops were affected during 1992-1994. To describe the impacts of the dis- ease, Arévalo et al. (2000) quantified its incidence and development in 11 coca fields in five regions in the valley. The coca plants sampled were 14-93 months old. Overall, the coca leaf yields were reduced by 74%. Disease incidence (the proportion of sampled plants that were diseased) ranged from 54% of 32-month- old plants to 79% of 93-month-old plants. The level of disease, measured as the area under the disease-progress curve, ranged from 5.67 on 39-month-old plants to 28.2 on 93-month-old plants (that is, the older plants were more diseased). Thus, the Peruvian F. oxysporum f.sp. erythroxyli epidemics of the 1980s and 1990s were highly devastating to coca production in the Huallaga Valley. No formal followup studies of the disease have been performed, but anec- dotal observations suggest that the coca wilt disease persists and affects coca leaf production in some portions of the valley. In some areas, however, cultiva- tion of coca has been moved to new fields to escape the disease (personal com- munication, E. Arévalo, Instituto de Cultivos Tropicales, November 16, 2010, to B. Bailey, USDA). Hence, the epidemic, even while inflicting severe losses, has not deterred coca production, which has continued even in the presence of spo- radic disease outbreaks.