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.
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).
Biological Control of Cannabis with Fusarium oxysporum f.sp. cannabis
Plants in the genus Cannabis, including C. sativa (fiber hemp and cannabis), 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 morphologically 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. oxysporum 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 polymorphisms may provide an alternative route for identification of these two formae 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 members 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 technique for the production of F. oxysporum f.sp. cannabis inoculum consisting of chlamydospores, which are suitable for soil application. Later, McCain and Noviello (1985) explored the feasibility of using F. oxysporum f.sp. cannabis (isolated 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 application. The findings led them to conclude that the disease caused by F. oxysporum 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 mycoherbicide (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 cannabis 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. Department 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 herbicide 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 symptoms (Darlington 1996). Pathogenicity and host-range experiments led the investigators 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 Huallaga 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 subpopulations of F. oxysporum f.sp. erythroxyli in the Huallaga Valley (Nelson et al. 1997). Later, Gracia-Garza et al. (1999) used RAPD and vegetative compatibility 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 natural 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 production 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 dispersal 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 efficacy, inoculum production and delivery, and persistence in the environment. No studies of F. oxysporum f.sp. erythroxyli for the control of coca have been published since 1999.
The overarching consideration in determining the feasibility of the proposed mycoherbicides is the ability of F. oxysporum f.sp. cannabis and F. oxysporum 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 (Lockdown), 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. Therefore, 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 assessments reported in the literature, including visual ratings and direct measurement of disease on individual plants and populations, help to quantify the destructiveness of the pathogens in controlled experiments, but they provide only an indirect 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 conference proceedings reported on the effectiveness of F. oxysporum f.sp. cannabis 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 collected in various regions of Kazakhstan. The details of the experimental methods used in those studies are presented in Tables 4-1 and 4-2.
TABLE 4-1 Greenhouse and Field Studies in Italy by McCain and Noviello (1985)
|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|
|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|
|Application rate and cultivars used||Vitulazio field trial: 10 g/m2; inoculated plots seeded with C. 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 required 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 planting.
McCain and Noviello reported that in field plots at Vitulazio, no Fusarium-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 fungusinfested 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 noninoculated control plots. At the highest inoculum level, 30 g/m2, only 4% and 14% of the cultivars CS and SF died.
TABLE 4-2 Greenhouse and Field Studies in Kazakhstan by Tiourebaev et al. (2001)
|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|
|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
|Application rate||12.5 g/m2 (plot size, 2 m2); inocula of the 25 isolates applied twice: 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 industrial 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.
The results of the study by McCain and Noviello confirm that F. oxysporum 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 authors’ 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 greenhouse 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. Tiourebaev 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. Isolate 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. oxysporum f.sp. cannabis as a pathogen of cannabis. However, the levels of disease, based on disease-severity and disease-incidence estimates, were low to moderate and led the authors to conclude that under field conditions “the infection 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 experiment 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, according to the authors, the “disease tapered off as cooler conditions prevailed”
(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 “microscopic analysis and colony morphology” (Tiourebaev et al. 2001). The authors assigned the name F. oxysporum f.sp. cannabis without completing Koch’s postulates 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 speciales 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 publications of McCain and Noviello (1985) and Tiourebaev et al. (2001) are not sufficient to draw conclusions about the feasibility of F. oxysporum f.sp. cannabis as a mycoherbicide.
Fusarium oxysporum f.sp. erythroxyli
The committee reviewed the available data on the efficacy of F. oxysporum 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 experiments in the field and in a growth chamber to assess the virulence of F. oxysporum f.sp. erythroxyli isolated from an Erythroxylum population growing in Hawaii. Bailey et al. (1997) performed three field trials in Hawaii with a ricealginate 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 transplanted, and the plants eventually died; however, no quantitative data were provided.
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
TABLE 4-3 Growth-Chamber and Field Studies by Sands et al. (1997)
|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|
|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 and rate||Subsurface method: inoculum (5.0 g/plot) deposited in 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 average 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, presumably 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 isolates 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. ) had died. F. oxysporum was isolated from the vascular tissues of symptomatic and asymptomatic plants from the fungus-infested soil and from symptomatic
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|
These results confirm F. oxysporum f.sp. erythroxyli as a virulent pathogen 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 fungaltreated 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 fungusfree soils, respectively) raises the question whether the naturally prevalent inoculum 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 compared 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 reported 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 disease.
The incidence of natural wilt of about 58% at the experimental site in Hawaii 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 problematic. Against such background disease levels, the increase in disease incidence resulting from the added inoculum cannot be ascertained on the basis of the collective 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 formulation germinated relatively uniformly in two greenhouse experiments and at least two field experiments when applied to the soil surface.” Thus, the ricealginate prill formulation appears to be a feasible and effective way to deliver the inoculum if it is deployed under conditions where adequate moisture is present.
Bailey et al. (1997), referring to Fusarium wilts in general, make the observation that the disease “becomes more severe with continuous cropping, and the pathogen often persists in infested fields for years after the crop is removed.” However, in the case of coca, anecdotal evidence from the Huallaga Valley of Peru, where a natural wilt epidemic caused by F. oxysporum f.sp. erythroxyli occurred in the 1990s, indicates that disease has not increased in severity during the last decade despite its sporadic outbreaks (Arévalo et al. 1994).
Mechanisms of Pathogenicity
There is no information on the mechanisms by which F. oxysporum f.sp. cannabis or F. oxysporum f.sp. erythroxyli causes disease in its host plants, but some generalizations can be drawn from what is known about vascular Fusarium wilts. F. oxysporum causes vascular wilt, as exhibited by leaf yellowing, loss of turgidity, necrosis, wilt, and plant death. Infection occurs when mycelium or germinating spores penetrate the roots of the plant, enter the xylem, and produce microconidia. Vascular vessels become clogged by the accumulation of mycelium, spores, and oxidation of breakdown products from enzymatic lysis. Toxins may be produced that cause vein clearing (loss of chlorophyll production along the veins), a reduction in photosynthesis, and tissue damage that leads to excessive water loss through transpiration. Symptoms typically start at the lower leaves and progress upward. Roots may also become infected and become stunted and rot (Desjardins 2006; Leslie and Summerell 2006; Garibaldi et al. in press).
Wilt-inducing F. oxysporum f. spp. are primarily soilborne, and epidemic development is predominantly monocyclic (only one disease cycle in a cropping season). Epidemics start from inoculum originating in plant debris in the soil in the form of chlamydospores (asexual, one-celled, thick-walled spores that can persist for a long time), macroconidia (the larger type of conidia or asexual spores formed by Fusarium spp.), and microconidia (the smaller type of conidia formed by Fusarium spp.). To a lesser extent, there is evidence that aerial inoculum (in the form of macroconidia and microconidia) is produced on infected stems and that more than one cycle of infection might be possible in a season (Timmer 1982; Gamliel et al. 1996; Katan et al. 1997; Rekah et al. 2000; Garibaldi et al. in press).
Facility, Equipment, and Technology for Large-Scale Manufacture
Large-scale production of mycoherbicides for commercial use is typically undertaken by industries that have microbial fermentation capability. Depending on the fungus, production would require submerged liquid fermentation, solidsubstrate fermentation, or a biphasic system in which the fungal biomass is first
produced in a liquid culture and then transferred to an organic or inorganic solid matrix for the production of spores or other desired propagules. If necessary, sophisticated approaches based on those principal systems could be developed (such as airlift fermentation). In general, the feasibility of adapting existing, readily available technology has been a strong incentive for industries to invest in producing mycoherbicides. The ability to produce the required amounts of mycoherbicides without major new investments or disruption of other commercial manufacturing commitments is an additional incentive. Several U.S. companies that specialize in the production of microbial biomass for pharmaceutical, food, biotechnology, and other industrial uses have the necessary technological capability and expertise to produce the proposed mycoherbicides (Bowers 1982; Churchill 1982).
Two mycoherbicides registered for use in North America (Collego and Sarritor) and an experimental mycoherbicide (CASST) are good examples for reviewing the industrial production processes that could be adapted to produce the required amounts of the proposed mycoherbicides. Collego (now registered as Lockdown and composed of Colletotrichum gloeosporioides f.sp. aeschynomene) is produced by submerged liquid fermentation. Sarritor (Sclerotinia minor) is produced by solid-substrate fermentation. CASST (Alternaria cassiae), a mycoherbicide that was industrially developed but not registered, for business reasons, was produced by using a biphasic method. The processes illustrated in Figure 4-1 can be modified in several ways to suit particular needs.
The proposed mycoherbicide agents F. oxysporum f.sp. cannabis and F. oxysporum f.sp. erythroxyli could be grown on a variety of solid substrates and liquid media to produce infective propagules, with emphasis on thick-walled chlamydospores that are more durable than conidia. Fermentation methods have been developed that specifically promote the production of chlamydospores (Hildebrand and McCain 1978; Hebbar et al. 1997; Connick et al. 1998). Although the available data on the proposed mycoherbicide strains provide useful leads, they are exploratory; any large-scale attempt at production must start with basic studies in an industrial setting to develop and optimize the production process. A few Fusarium species are currently mass-produced by industrial fermentation processes for production of various commercial products, such as gibberellic acid, zearalenone, and edible mycoprotein (Waites et al. 2001).
In addition, Elzein and Kroschel (2004) and Watson et al. (2007) have developed laboratory-scale production methods for F. oxysporum strains for the control of the parasitic weed striga (Striga hermonthica). Collectively, experience with these fermentation methods could be used to guide the development of the proposed Fusarium mycoherbicides. Typically, industrial studies are needed to determine the choice of fermentation method (liquid, solid, or biphasic), the type of propagules (mycelium, asexual spore [conidium], or resting spore or structure [chlamydospore or microsclerotia]), the formulation (aqueous liquid concentrate, emulsion, dust, pellet, food-grain-based, seed-based, and so on), the intended delivery method (aerial or ground-based; directed or dispersed), and expected efficacy, shelf-life, and handling specifications of the product.
The committee reviewed studies by Hildebrand and McCain (1978), McCain and Noviello (1985), and Tiourebaev et al. (2001) to assess the materials and methods used for the production of F. oxysporum f.sp. cannabis inoculum for field trials. Studies by Sands et al. (1997) and Hebbar et al. (1997) were reviewed to assess the production of F. oxysporum f.sp. erythroxyli inoculum for field trials. The available data on the media and methods used and the amounts of inoculum produced are given in Table 4-5. These data were used to estimate the amount of inoculum that might be needed to treat illicit cannabis and coca crops.
Feasibility of Large-Scale Production
To estimate the likely amounts of the mycoherbicide products that might be required for a hypothetical operational drug-crop control program worldwide, the committee made calculations (Table 4-6) based on published data on the
amounts of inoculum produced for use in field trials (see Table 4-5). However, the committee cautions that the published data are at best a guide because they are rudimentary in scope and based on arbitrary choices of materials and methods. Therefore, the actual amounts needed can only be determined by testing different formulations under field conditions. Such testing is best done in collaboration with an industrial producer of fungal-based products. The actual amounts required to control the drug crops may or may not be feasible to produce due to cost and/or technical limitations.
Adjuvants and Formulation
Although there are exceptions, the registered mycoherbicides are generally applied without adjuvants, substances added with but separate in function from the mycoherbicide formulation and intended to improve the mycoherbicide’s effects, such as spreaders-stickers, infection aids, and virulence enhancers. For example, a sugar (high-fructose corn syrup) is added to the preparation of Collego (Lockdown) for regulating rehydration of dry spores. The sugar enables a slow and gradual hydration of the spores, which is critical for maintaining spore viability during application (Churchill 1982).
Formulation is crucial not only to guarantee mycoherbicide performance but also to improve its efficacy, shelf life, ease of handling, and application. A range of formulations—from wettable powder (Collego/Lockdown) to liquid concentrate (DeVine) to paste (Chontrol) and granules (Sarritor), have been used commercially. To avoid drift and unintentional exposure to a harsh environment, it is unlikely that the proposed F. oxysporum mycoherbicides would be sprayed in water or dusted as a fungal powder. Solid media, such as translucent or lightcolored alginate formulations, would help to protect the live pathogen propagules from desiccation and ultraviolet radiation. Such formulations could include a nutrient base—such as amino acids, sugars, and some fatty acids—for initial growth, survival, and establishment of the pathogens at the target site (Hildebrand and McCain 1978; Gracia-Garza et al. 1998; Hebbar et al. 1999).
A review of published data on the proposed mycoherbicide strains indicates that several formulations were developed and tested in the field although only on a small scale. The committee reviewed studies by Hildebrand and McCain (1978), McCain and Noviello (1985), and Tiourebaev et al. (2001) on F. oxysporum f.sp. cannabis and by Hebbar et al. (1997, 1999), Sands et al (1997), Bailey et al. (1997, 1998), and Connick et al. (1998) on F. oxysporum f.sp. erythroxyli. The formulations tested and their viability or efficacy are summarized in Table 4-7.
The study by McCain and Noviello (1985) evaluated the feasibility of producing stable and efficacious inoculum composed of straw and soybean meal colonized by the fungus, but the inoculum was not characterized in terms of
TABLE 4-5 Media and Methods Used for Production of Fusarium oxysporum f.sp. cannabis and Fusarium oxysporum f.sp. erythroxyli for Field Trials
|F. oxysporum f. sp. cannabis|
|Hildebrand and McCain 1978|
|Extracts of various plant products (prepared by autoclaving 75 g of a product in 50 mL of distilled water at 212° C for 15 min and decanting supernatant liquid) inoculated with 0.1 mL of aqueous conidia + mycelium suspension and incubated for 2 wk||The following amounts of chlamydospores produced in each substrate tested:
Extract of alfalfa straw: 192 × 104 or ~ 3.8 × 104/mL of medium
Extract of cottonseed meal: 90 × 104 or ~ 1.8 × 104/mL of medium
Extract of soybean meal: 24 × 104 or ~ 0.5 × 104/mL of medium
|Barley straw (800 g), 2 L distilled water plus 160 g of soybean meal, alfalfa straw, or cottonseed meal or glycine- succinate- NaNO3 solution inoculated with the fungus, incubated for 3 wk in 2- to 3- L glass flasks at room temperature, removed from the flasks, air dried for 2 wk, and added to the soil at 10 g/1,000 g of soil; number of propagules in soil determined after 5 wk||Per gram of fungus- colonized soil:
Barley straw plus cottonseed meal: 3.5 × 104
Barley straw plus alfalfa: 2.7 × 104
Barely straw plus succinate- glycine- NaNO3 mixture: 2.5 × 104
Barley straw plus soybean meal: 1.46 × 104
|McCain and Noviello 1985|
|F. oxysporum f. sp. cannabis cultured on twice- autoclaved barley straw and soybean meal (oil extracted) mixture for 2-4 wk and air dried for 5-7 d||Inoculum composed of particles of straw and soybean meal with fungal mycelium, conidia, and chlamydospores
Number of fungal propagules per gram of colonized and air- dried straw and soybean meal not stated in paper
|Tiourebaev et al. 2001|
|Fungus cultured in 100 mL of potato dextrose broth in 250- mL flasks for 2-3 d with constant agitation at 200 rpm
Cultures homogenized by stirring and then suspended in a membrane-stabilizing solution composed of 1,000 mM sucrose, 500 mM sorbitol, and 10% tryptone
Homogenized culture mixed with birch sawdust, wheat seeds, or oat seeds and then used as inoculum for experiments
|Amount of conidia per gram of formulations not stated in paper|
|F. oxysporum f. sp. erythroxyli|
|Sands et al. 1997|
|Potato dextrose broth culture of F. oxysporum f. sp. erythroxyli mixed with sterilized, dehulled, milled rice grains and inoculated with 3.3 × 105 conidia/g of rice
Treated rice air- dried for 12- 24 h and then mixed with steamed greenhouse soil (1:100, wt/wt); rice- soil mixture moistened and fungus allowed to grow throughout the soil for 1-3 wk
|Type and amount of infective propagules produced on rice- soil mixture not stated in paper|
|Hebbar et al. 1997|
|Used a bench- top fermentation with an aqueous extract of soybean- hull fiber that had been autoclaved, filtered, and poured into a 2.5- L fermentor vessel; three fermentation conditions tested: (1) T1 = sparged (bubbled) from 0 to 14 d, (2) T2 = nonsparged from 0 to 14 d, (3) T3 = nonsparged from 0 to 14 d and pH raised after 4 d and maintained at 9.0- 10.0 For fermentation in 20- L carboys, autoclaved nonfiltered soybean- hull fiber used as medium||Liquid- fermentation method increased chlamydospore yield (6.3 × 106 chlamydospores/mL) after fermentation for 14 d, with biomass viable counts of 1 × 108 propagules/g of air- dried biomass Methods increased chlamydospore yields and reduced time needed for their production from 5 wk to 2 wk|
|Connick et al. 1998|
|Biomass of F. oxysporum f. sp. erythroxyli strain EN- 4 produced by method of Hebbar et al. (1997) with aqueous extract of soybean fiber in medium||3 × 107 CFU/mL produced with this method Conidia then air- dried and used in laboratory- scale Pesta formulations (containing 5.5 × 107 CFU/g) and twin- screw extrusion formulation (containing 1.3 × 108 CFU/g); biomass used in Pesta formulation composed mostly of chlamydospores|
TABLE 4-6 Estimated Amounts of Proposed Fusarium oxysporum Mycoherbicides Needed for Single Application Against Illicit Cannabis and Coca Crops Worldwide
|Mycoherbicide—Target Drug Crop||Reference||Amount of Inoculum Reportedly Used in Field Trialsa||Amount of Mycoherbicide Needed for Each Application over potential worldwide area,b metric tons||Amount of Mycoherbicide Needed for Each Application, kg/ha|
|F. oxysporum f.sp. cannabis—cannabis||McCain and Noviello 1985||1-30 g/2, inoculum composed of fungus-colonized straw and soybean mealc||2.000-192.540d||10-300|
|F. oxysporum f.sp. cannabis—cannabis||Tiourebaev et al. 20011||2.5 g/m2, inoculum composed of liquid fungal culture at -33% by weight mixed with birch sawdust, wheat seeds, or oat seeds and air-dried: two applications of inoculum 1 mo apart||25-80.2 million||125|
|F. oxysporum f.sp.||Sands etal. 1997||5 g /0.3 m2. inoculum composed of fungus-colonized millet seeds:||26.996||170|
|eiythroxyli-coca||proportion of fungus to millet seed not specified|
|F. oxysporum f.sp.||Bailey etaL 1997||33.6 kg/ ha. inoculum composed of alginate prill formulation||5,336||34|
|eiythroxyli-coca||containing 1x106 to 5.3 x 106 CFU|
|F. oxysporum f.sp.||Bailey etaL 1998 eiythroxyli-coca||33.6 kg/ha. inoculum composed of rice-alginate prill, wheat flour-kaolin (Pesta). and rice flour-wheat flour (C6). each containing 1 x 106 to 5.3 x 106CFU||5,336||34|
aAmounts of inoculum used in field experiments were not aimed at defining minimum inoculum quantity needed for effectiveness; tliis remains to be determined with actual mycoherbicide products.
bEstimated potential worldwide target area for mycoherbicide use in hectares is based on UN Office on Drugs and Crime’s most recent estimate of total area under cannabis and coca cultivation worldwide (UNODC 2010a): 200.000-64LS00 ha and 158.S00 ha. respectively. Calculations provided on the basis of potential worldwide target area to indicate 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.
cLower inoculum level reported to be less effective than higher level: nonetheless, both figures are used to indicate low and high estimates of inoculum needed.
dFor a perspective, a familiar automobile, such as Toyota Prius. weighs 1.33 metric tons. Low-end amount of inoculum needed for single application would approximate the weight of 1.504 Prius cars.
TABLE 4-7 Developed and Tested Formulations of Fusarium oxysporum f.sp. cannabis and Fusarium oxysporum f.sp. erythroxyli
|Formulation||Viability and Efficacy|
|Fusarium oxysporum f. sp. cannabis|
|Hildebrand and McCain (1978)|
|Fungus + barley straw + soybean meal;
fungus + barley straw + alfalfa straw;
fungus + barley straw + cottonseed meal;
fungus + barley straw + glycine-succinate-NaNO3 solution;
all inoculated straw-substrate mixtures air-dried for 2 wk)
|Air-dried inoculum produced on all straw-substrate mixtures and stored in plastic bags at room temperature reportedly remained efficacious for 6 mo; seedlings inoculated with formulations stored for 6 mo died within 18-20 d after inoculation; loss in efficacy noted for formulations stored for 9 mo and 12 m; seedlings inoculated with formulations stored for 9 mo and 12 mo died after 24 and 30 d after inoculation, respectively|
|McCain and Noviello (1985)|
|Fungus + barley straw and soybean meal (oil-extracted), incubated for 2-4 wk and air-dried for 5-7 d||Inoculum (particles of straw and soybean meal with fungal mycelium, conidia, and chlamydospores) stored at 20-22° C; no “appreciable loss” of viability observed after a year of storage at 20-22° C or -10° C|
|Tiourebaev et al. (2001)|
|Fungus suspended in solution composed of 1,000 mM sucrose, 500 mM sorbitol, and 10% tryptone + carrier (wheat seed, oat seed, or birch sawdust soaked in yeast extract plus 1 M citric acid (pH, 5.5) solution for 30 min and then autoclaved); inoculated carrier incubated at room temperature for 2 d (to allow mycelial growth) and then air-dried at ambient temperature for about 5 h and stored at 5° C||Wheat seed, oat seed, and birch-sawdust formulations used in greenhouse and field experiments; wheat and oat formulations “ heavily predated” by insects, lizards, and rodents, and this reduced their efficacy in the field|
|Fusarium oxysporum f. sp. erythroxyli|
|Hebbar et al. (1997)|
|Bench-top fermentation: fungus + aqueous extract of soybean-hull fiber (autoclaved, filtered, and poured into a 2.5-L fermentor vessel); fermented for 14 d; fermentation in 20-L carboys; fungus + soybean-hull fiber (autoclaved, nonfiltered)||After 14 d of fermentation (bench-top), mostly microconidia and chlamydospores produced; average number of viable propagules (determined on potato-dextrose agar plates) was 2.5 × 107 propagules/mL; inoculum produced with this fermentation method not tested in greenhouse or field for efficacy against coca plants|
|Formulation||Viability and Efficacy|
|Sands et al. (1997)|
|Fungal conidia (from potato-dextrose broth culture) + rice grains (autoclaved, dehulled, milled); conidia + rice grain mixture air-dried for 12-24 h and mixed uniformly with steamed greenhouse soil (1:100, w/w)||Rice grain-soil formulation used in greenhouse and field trials where disease symptoms (leaf drop and plant death) were observed|
|Formulation||Viability and Efficacy|
|Bailey et al. (1998)|
|Rice alginate prill; C6 (rice flour + wheat flour); Pesta (wheat flour + kaolin)||All three formulations enhanced F. oxysporum f. sp. erythroxyli EN-4 strain population in soil (greenhouse and field experiments)|
|Bailey et al. (1997)|
|Rice alginate prill formulation||In greenhouse tests, application of viable rice alginate prill formulation at 33.6 kg/ha significantly increased soil fungal population (four soil types tested); population remained 1-2 log units higher in top 5.1 cm of soil than in lower 5.1-10.2 cm throughout 7-wk experiment|
|Connick et al. (1998)|
|Pesta formulation (32 g semolina, 5 g kaolin, 3 g fungal biomass, 23 and mL deionized water); twin-screw extrusion formulation (930 g semolina, 232 g kaolin, 112-150 g dried fungal biomass, and 500 mL deionized water, blended together in a food processor and then dried to water activity of 0.24-0.38)||Viability of Pesta formulation tested in laboratory with water agar plates; percentage germination of F. oxysporum f. sp. erythroxyli determined at 4-or 8-wk intervals for 52 wk; F. oxysporum f. sp. erythroxyli in Pesta granules had shelf life of at least 1 yr when stored at 35° C (0.12 aw) and 2 yr if stored at 25° C (0.12 and 0.33 aw); granules made with twin-screw extrusion method retained 100% of its CFU/g after extrusion and drying at 35° C, and 77% of CFU/g after drying at 50° C|
|aw= water activity.|
colony-forming units per gram of soil or the proportion of fungal biomass relative to the substrate. Such information is essential for standardization and assurance of the efficacy of the formulation. McCain and Noviello also mentioned the use of other animal feeds to produce the inoculum but did not provide further details. Finally, the application method that they used to infest the field soil, distribution of the inoculum by hand and its incorporation into the top 10 cm of the soil, is not practical for field operations to control drug crops.
There are two problems with the food-based formulations used by Tiourebaev et al. (2001). First, no data were presented on the characteristics of the formulations, such as the number of colony-forming units. Second, the amount of mycoherbicide needed, 125 kg/ha, applied twice in field trials, is too large to be feasible (see Table 4-6). Another serious limitation was the loss of inoculum through predation by fauna, which raises the prospect that formulation with food materials might not be an effective method of delivery of mycoherbicides.
The method of delivery of a mycoherbicide is dictated by the biology of the pathogen (whether soilborne or aerial); its mode of action, for example, whether it causes a foliar blight or vascular wilt or infects through wounds; formulation type (liquid or dry); the feasibility of the delivery method; and the desired objective, for example, to kill the target plant population or only to establish disease in it. How the target plant is grown also is likely to dictate the method of delivery; F. oxysporum f.sp. erythroxyli would probably be applied from the air, while F. oxysporum f.sp. cannabis might be applied with a ground rig because the plantings often are placed under the canopy of trees, for example, in the southwestern United States. The currently registered mycoherbicides are applied aerially from fixed-wing aircraft (Collego/Lockdown) or backpack or tractor-based sprayer (DeVine), by painting cut stumps (Chontrol), by spreading with a fertilizer spreader or by hand (Sarritor), and by other methods. With mycoherbicides used in agriculture and forestry, the prevalent application and cropping practices largely dictate the method of delivery, and any extra cost and effort needed to apply mycoherbicides with novel tools or methods would be a disincentive.
Although on-ground application of mycoherbicides for cannabis and coca is possible, from a tactical standpoint only aerial application seems feasible for application over areas where growers are uncooperative and possibly hostile. However, aerial application of dry formulations—that is, in powder or pellet forms—might not be effective with a soilborne pathogen, such as F. oxysporum. Furthermore, aerial application of a dry formulation is likely to result in a nonuniform, discontinuous placement of the inoculum over the target area and thus limit opportunities for plant-pathogen contacts.
Sands et al. (2002) developed a patented technology to deliver a soilborne fungus, such as F. oxysporum, by aerial dispersion. By dispersing live seeds of
cannabis, tall fescue (Festuca arundinacea), bluebunch wheatgrass (Agropyron spicatum), and tomato treated with a coating agent (carboxymethyl cellulose, methyl cellulose, Mycotech oil, or potato-dextrose broth) and fungal spores, they have demonstrated soil penetration of the fungus and its establishment in the root zone. The germinating seed not only carries the fungal inoculum into the soil but enables the active concentration of a mycoherbicide fungus, such as F. oxysporum f.sp. cannabis, to increase in the root zone of the carrier plant. This method of delivering the inoculum has been tested in two small-scale field trials in Kazakhstan, where the formulation of cannabis seeds coated with F. oxysporum f.sp. cannabis yielded higher disease incidence than did a birchsawdust formulation or a control without inoculum. This novel method offers an intriguing possibility for delivering mycoherbicides that contain soilborne pathogens, but it requires further testing on a large scale to determine its utility as a method for delivering the proposed mycoherbicides.
The committee reviewed published methods for applying F. oxysporum f.sp. cannabis and F. oxysporum f.sp. erythroxyli used in greenhouse and field tests conducted by McCain and Noviello (1985), Tiourebaev et al. (2001), Sands et al. (1997, 2002), and Bailey et al. (1997, 1998). It also considered an innovative method of mycoherbicide delivery: deploying (from a cargo aircraft) seeds that have been colonized by the mycoherbicide fungus and coated with controlled-release polymers, which are meant to protect the biological agent until optimal environmental conditions occur (Nowak and Eusebi 2010). This method is interesting but so far has not been tested in the field with any mycoherbicide fungi. As for tools or implements for delivery of mycoherbicides, little has been done; dry formulations were spread by hand over the soil surface or incorporated into the soil below the surface, presumably with an implement like a hoe. The rates of application varied from one study to another; in some studies, the method, equipment, or tools used for delivery of inoculum to the soil were not described in enough detail to draw conclusions. Details of the delivery methods tested are summarized in Table 4-8.
Assessment of Performance
The performance of registered mycoherbicides is usually assessed as a percentage of control of the target weed (based on the number of plants in a population killed); as reduction in competition for space, nutrients, water, and light from the weed (collectively termed reduction in weed interference); or as alleviation of yield loss due to the weed. Assessment of efficacy of
F. oxysporum f.sp. cannabis was based on plant mortality (McCain and Noviello 1985) or on a disease-severity scale of 1-5, in which 1 = healthy plant, 2 = wilting of lower leaves, 3 = wilting of 25-50% of leaves, 4 = wilting of over 50% of leaves, and 5 = plant death (Tiourebaev et al. 2001). Assessment of efficacy of F. oxysporum f.sp. erythroxyli was based on a disease-severity scale of 0-2, where 0 = no disease, 1 = plant wilting, and 2 = plant death (Sands et al. 1997). In field trials, Bailey et al. (1997, 1998) used a disease-severity scale of 0-2, where 0 = plant asymptomatic, 1 = symptomatic plant that was defoliating, and 2 = plant death. Such assessments are typically used in academic research and are possible on a small scale when growers are cooperative; that is unlikely to be the case with illicit-drug crops.
TABLE 4-8 Methods of Delivering Fusarium oxysporum f.sp. cannabis and Fusarium oxysporum f.sp. erythroxyli in Greenhouse and Field Experiments
|F. oxysporum f.sp. cannabis|
|McCain and Noviello 1985||In field experiments: inoculum (barley straw and soybean meal particles with fungal mycelium, conidia, and chlamydospores) distributed by hand and mixed with top 10 cm of soil
In soil survival experiments (in the field): inoculum incorporated into the top 7.5 cm of soil at 1 g/m2 and 10 g/m2
|Tiourebaev et al. 2001||In greenhouse experiments: 0.5 g of carrier (inoculated with the fungus) scattered on the surface of soil in a 10-cm2 pot; equipment or tools used to scatter the inoculum not stated in the paper
In field experiments: inoculum (mixed with carrier) broadcast at 12.5 g/m2
|F. oxysporum f.sp. erythroxyli|
|Sands et al. 1997||In growth-chamber experiments: millet seeds colonized by fungus inserted into 10-cm-deep holes in potted soil
In field experiments: (1) millet seeds colonized by fungus deposited into a 2-cm-deep V-shaped trench and covered with 2-3 cm of soil (5.0 g millet seeds per plot) or (2) millet seeds colonized by fungus applied onto soil surface at 5.0 g/plot
|Bailey et al. 1997||In greenhouse and field experiments: Rice alginate prill formulation applied to soil surface at 33.6 kg/ha; method of applying to soil surface not specified|
|Bailey et al. 1998||Various formulations applied to soil surface at 33.6 kg/ha; method of applying to soil surface not specified|
In the case of the proposed mycoherbicides, “acceptable performance” is inadequately defined. As noted earlier, complete eradication (100% of the plants killed) is an unrealistic objective, but high disease incidence that results in high yield losses might be possible, such as over 50% loss, as has been reported in the natural epidemics of F. oxysporum f.sp. erythroxyli in Peru in the 1990s. In the absence of quick, clear-cut, and measurable results, it might be impossible to assess the performance of the mycoherbicides in operational drug-control programs. Inconsistency of performance by the mycoherbicide strains due to unfavorable climatic conditions, improper timing of application, incorrect method of application, and other factors could render assessments difficult, subjective, and unreliable. On-the-ground scouting and grower surveys could provide reliable estimates, but the safety of the personnel assigned to this task must be considered. Estimates from aerial surveillance probably are not feasible for cannabis and coca, which often are grown as an understory crop or in the midst of natural vegetation, such as rain forests, woodlands, and parklands. Thus, the lack of tools and methods for assessing the performance of mycoherbicides in a field drug-crop control situation does not allow an informed choice about using mycoherbicides to control illicit-drug crops.
The persistence of the proposed F. oxysporum mycoherbicides in the environment is important to determine whether their population density would remain high enough and last long enough to infect the target crops and whether they could survive in the soil and organic matter at levels necessary to affect later plantings of the crop. Another consideration is whether the mycoherbicide strains pose risks to nontarget organisms after release. If such risks occur, the prolonged persistence of the strains would be a disadvantage rather than an advantage. The ability to persist in the environment is an important consideration in proposing the use of a mycoherbicide strain. Monitoring for persistence of registered mycoherbicides has been based on molecular characteristics of the fungus (Dauch et al. 2003; Ditmore et al. 2008).
In general, the efficacy and survival of fungal pathogens depends on environmental conditions, such as temperature, soil composition, moisture (dew period, relative humidity, and rainfall for foliar pathogens; soil moisture and water potential for root pathogens), and ultraviolet radiation. Mycoherbicides are typically formulated to enhance pathogen survival after application to ensure successful infection and establishment on the target plant and possibly to allow for the production of secondary inoculum to keep the disease going during the season or to provide inoculum for the next season. Longer-term survival would depend on favorable environmental conditions. Other factors that influence survival include geographic considerations, the movement of the pathogen outside the application area, the ability of the pathogen to survive on other plant hosts, and the nature and composition of the resident soil biota.
Geographic and Climatic Considerations
Only one study that provides data on the environmental conditions that affect the establishment of F. oxysporum f.sp. cannabis on inoculated plants was found (Tiourebaev et al. 2001). Infection of cannabis began to appear in field trials conducted in Kazakhstan in May and June, when temperatures were 20-30°C. Disease was highest during late August and September and then declined as “cooler conditions prevailed”; this suggests that the host plant continued to grow while disease development was slowed or arrested at cooler temperatures. However, no quantitative data on the pathogen population size were reported, so it is unknown whether the decreased disease progression was due to decreased numbers of the pathogen. No studies have specifically investigated the effects of different environmental conditions (such as soil composition, temperature, and moisture) on the persistence of F. oxysporum f.sp. cannabis, but any single strain may have a narrower set of climatic boundaries than cannabis, especially inasmuch as cannabis is grown in a wide variety of environmental conditions (see Chapter 3).
Three studies have evaluated the effects of environmental factors—such as soil type, moisture (soil matric potential and relative humidity), and temperature—on the proliferation and survival of F. oxysporum f.sp. erythroxyli (see Tables 4-9 and 4-10 for details). Fravel et al. (1996) found that F. oxysporum f.sp. erythroxyli (strain EN4-FT) can survive in different soil types, at temperatures of 10-35°C, and under stable moisture conditions (such as relative humidity of about 80-97%, regular misting, or irrigation). However, some of the soils tested appear to have had a “suppressive” effect on the pathogen’s survival. Followup studies with autoclaved and nonautoclaved soils indicated that native microbial communities in the soils inhibit the survival of the pathogen.
Studies by Bailey et al. (1997, 1998) showed that under some field conditions F. oxysporum f.sp. erythroxyli (EN-4 strain) can persist for at least 6 or 7 months in the soil with a decline in density over that period. The three formulations tested performed similarly in enhancing soil colonization. Most of the colonization occurred in the top 1-5 cm of soil, and the fungus was shown to colonize coca roots. Moisture was controlled during the first 10-14 days after treatment by drip irrigation, presumably to provide favorable conditions for infection. Enhancement of the F. oxysporum f.sp. erythroxyli population in soil appeared to be greater when spores were applied to fields previously planted with coca.
Greenhouse and small field tests indicated that F. oxysporum f.sp. erythroxyli EN-4 can grow and survive at temperatures ranging from 10 to 35°C and that survival is poor below 10°C and above 40°C (Fravel et al. 1996; Bailey et al. 1997, 1998). Moreover, high soil moisture is needed for its initial establishment.
|Test Factors and Methods||Results|
|-10, -100, and -500 kPa 10 prills/plate Soil types tested: Galestown gravelly loamy sand (GGLS) Hatbro loamy sand (HLS) Red clay subsoil (RC) Hawaiian clay (HCL)||Matric potential of soils affected survival of F. oxysporum f.sp. erythroxyli; density in HCL soil greatest at -100 kPa, but density in GGLS soil greatest at -10 kPa; in all soils, survival significantly less at -500 kPa (drier soil) than at -100 or -10 kPa (moister soils) Population density of F. oxysporum f.sp. erythroxyli greatest in HCL soil and least in HLS soil|
|Soil samples taken after 1 wk Treatments replicated three times Experiment repeated three times|
|10, 18, 25, 32, and 40°C Soil types tested: GGLS, HLS, and RC Soil samples taken after 1, 5, 9, 13, and 17 wk 10 prills/petri dish (75 petri dishes/soil type) Treatments were replicated three times Experiment repeated once||F. oxysporum f.sp. erythroxyli survival greatest at 10-32°C; no significant differences in survival between soils tested Population density 103-105 CFU/g after 1 wk, depending on temperature, and declined to 0-104 after 17 wk|
|Alternating 15C° and 25°C every 12 h Alternating 25°C and 35°C every 12 h Soil types tested: GGLS, HLS, and RC Soil samples taken after 1, 5, 9, 13, and 17 wk 10 prills/petri dish (75 petri dishes/soil type) Treatments replicated three times Experiment repeated once||F. oxysporum f.sp. erythroxyli survival did not differ between temperature regimes, but persistence was affected by time and soil type Initial population density was 104-106 CFU/g and declined over study; decline most rapid in RC and HLS soils, especially for 25-35°C regime, in which populations fell below detection levels after 17 wk|
aFor all tests, alginate prill formulation (105 CFU/prill) was used and 10 prills were added to 25 g of soil. Treatments replicated three times. Source: Adapted from Fravel et al. 1996.
TABLE 4-10 Greenhouse and Field Studies of Effects of Environmental Factors on Survival of Fusarium oxysporum f.sp. erythroxyli
|Bailey et al. 1997|
|Formulation and application||Rice alginate prill F. oxysporum f.sp. eiythroxyli strain EX-4 (primarily chlamydospores) Application rate. 3.3 g/ha||Rice alginate prill F. oxysporum f.sp. erythroxyli strain EN-4 (primarily chlamydospores) Application rate. 33 g/ha|
|Coca species and plant age||E. coca seedlings||Experiment 1: E. coca, 15 mo|
|Experiment 2: E. coca, 16 mo|
|Experiment 3: E. coca, 17 mo|
|Environmental conditions||Four soil types tested: Galestown gravelly loamy sand Hatboro loamy sand Red clay subsoil Hawaiian clay||Kauai. Hawaii 17-21°C (average minimum). 23-27°C (average maximum). >80% to <97% relative humidity, drip irrigation for first 10 d|
|29°C. 14-h dayjlight: 22°C. 12-h night for 7 wk Soils watered once per week, misted daily||Experiments 1 and 2: fields previously grown with coca for 7 yr or more Experiment 3: field not previously grown with coca|
|Soil sampling method||Samples taken 0, 1, 2, 4, and 7 wk after treatment||experiments 1 and 2: single samples taken from each plot 0. 2o. 56. and 229 d after treatment|
|EN-4 distinguished from background Fusarium on basis of colony morphology and distinct orange color||Experiment 3: single samples taken 0 and 200 d after treatment (technical problems prevented collection of data between these days) Samples taken from top 7.6 cm of soil and at 7.6-15.2 cm|
• EX-4 population was significantly greater than control, similarly in all four soil types
• EN-4 population significantly greater in soil than control
• EN-4 maximum population of 4.265 CFU/g after 2 wk. declined to 1.060 CFU. 2 over next 5 wk
• Population highest in top 7.6cm ot soil (maximum. 534 CFU/g after 1 mo. declined to 95 CFU/g over next 6 mo)
|• EN-4 population highest in top 5.1 cm of soil||• At soil depths of 7.6-15.2 cm. EX-4 populations low (maximum. 22 CFU. 2 after 1 mo)|
|• EN-4 in control samples averaged less than 4 CFU/g for entire study period||• EN-4 in control plots below 10 CFU/g for entire study period|
• EN-4 population highest in top 7.6 cm of soil (maximum. 9.015 CFU/’g after 1 mo. declined to 114 CFU/g over next 6 mo: not detected after 7 mo)
• At soil depths of 7.6-15.2 cm. EX-4 populations low (maximum. 36 CFUg after 2 mo)
• EN-4 in control plots below 10 CFUv g for entire study period
Data collected only at 6 mo because of technical problems
EN-4 population 5S CFU/g in top 7.6 cm ot soiL
• EN-4 not detected at 7.6-15.2 cm of soil
|Replication||Three replications of each soil-treatment combination Experiment repeated||Experiment 1: five replications of each treatment
Experiment 2: four replications of each treatment
Experiment 3: five replications of each treatment
|Bailey et al. 1998|
|Formulation and application||Rice alginate prill, wheat-flour kaolin, and rice and wheat-flow mixture F. oxysporum f.sp. erythroxyli strain EN-4 Application rate. 33 g/ha||Rice alginate prill, wheat-flour kaolin, and rice and wheat-flour mixture F. oxysporum f.sp. eiythroxyli strain EN-4 Application rate. 33 g/ha|
|Coca species and plant age||E. coca seedlings||Experiment 1: E. coca, 9 mo old
Experiment 2: E. coca, 5 mo old
Experiment 3: E. coca, 7 mo old
|Environmental conditions||Galestown gravelly loamy sand 29°C 14-h daylight. 22°C 10-h night for 6 \vk||Kauai. Hawaii Drip irrigation for first 14 d after application Experiment 1: field not previously grown with coca: temperature and moisture not specified Experiment 2: field previously grown with coca for 7 yr or more: 24°C (soil). 21°C (ail-). >80% humidity over fust 10 d of treatment Experiment 2: field previously grown with coca for 7 yr or more: 23°C (soil). 20°C (air). <97% humidity over first 10 d of treatment|
|Soil sampling method||Two samples taken 1. 4. and 6 wk after treatment
Three samples taken 2 and 6 wk after treatment
|Experiment 1: sampling method not specified
Experiment 2: samples taken 0, 10, 33, 60, and 232 d after treatment
Experiment 3: samples taken at 0, 11, 28, 62, and 213 d after
Samples taken from top 1 cm of soil and at 7.6-15.2 cm treatment
|Results||• All formulations resulted in greater populations of EX-4 in soil than in untreated controls: rice alginate prill had lowest increase
• EX-4 populations highest in top 1 cm of soil (mean. 5.587 CFU/g)
• At depths of 5.1-10.2 cm. EN-4 populations low (mean. 20 CFU/g)
• After 2-5 wk. mean EX-4 populations declined from 31.804 CFU/g to 5.587 CFU/g
|Experiment 1: No data on EX-4 populations reported
• All three formulations resulted in greater populations of EX-4 in top 1 cm of soil than in control (mean maximum. 15.135 CFU g.within 10 d. declined to 29 CFU g at 232 d)
• No significant difference in EX-4 populations between soil depths of 7.6-15.2 cm and controls and EX-4 not detected at end of study
• All three formulations resulted in greater populations of EX-4 in top 1 cm of soil and at 7.6-15.2 cm than in untreated controls
• Maximum mean of 79.432 CFU g in top 1 cm of soil was about five times greater than in Experiment 2
• EN-4 barely detectable at depths of 7.6-15.2 cm after 213d
|Replication||Four replications ot each treatment
Experiment was repeated
|Experiment 1: four replications of each treatment
Experiment 2: six replications of each treatment
Experiment 3: six replications of each treatment
Transmission and Spread
The potential pathways by which spores and other propagules from a particular application site might move via physical transport mechanisms are illustrated in Chapter 2 (see Figure 2-1). In general, dispersal of the proposed mycoherbicides 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 infected plant tissues and seeds (Gambogi 1983; Gracia-Garza et al. 1999; Rekah et al. 2001). However, additional evidence indicates that under humid conditions, 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 application site by several vectors, including humans (transporting plant material or infested seeds), the hides or fur of mammals (such as pack animals), the droppings of birds, or the surface of insects. Long-distance transport via waterways is a potential route inasmuch as viable and infectious propagules of F. oxysporum 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 unintended spread beyond target areas in that farmers and traders could carry infected materials throughout a region or even into new areas, as has been proposed 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
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 longterm 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 individuals 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. oxysporum formae speciales and other fungi suggest that the presence of competitor 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 responsible 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.
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.
oxysporum f.sp. erythroxyli could survive on plants other than coca and on decaying 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. oxysporum 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 conclusions beyond this generalization.
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 organisms. Consideration is given to the issues specified in the committee’s statement 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 proceedings 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 potato) 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
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 experiment, 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)
Cucumus sativus (cucumber)
Daucus carota (wild carrot)
Festuca arundinaceae (tall fescue; grass)
Gossypium hirsutum (cotton)
Hordeum vulgare (barley)
Lycopersicon esculentum (tomato)
Melilotus indica (sweet clover; herb)
Phaseolus vulgaris (kidney bean)
Pisum sativum (garden pea)
Raphanus sativus (radish)
Triticum aestivum (wheat)
Zea mays (corn)
Lycopersicon esculentum (tomato)
Phaseolus vulgaris (kidney bean)
Solanum tuberosum (potato)
Triticum aestivum (wheat)
Zea mays (corn)
Agropyrum pectiniforme (crested wheatgrass)
Alhagi pseudalhagi (camelthorn; legume)
Artemisia vulgaris (common wormwood; herbaceous perennial)
Atriplex alba (lambsquarters goosefoot; shrub)
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)
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. Erythroxylum 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 alternative 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 products (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 Czechoslovakia, India, Iran, Mexico, Nepal, Pakistan, Poland, South Africa, Thailand, and
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 provided 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 pathogen 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 members 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. Biosynthesis of secondary metabolites often begins with components derived from primary 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 exposures to produce any observable symptoms.
Some members of the genus Fusarium produce toxic secondary metabolites (Marasas et al. 1984; Desjardins 2006). Table 4-13 presents a summary of the classes of mycotoxins reportedly produced by F. oxysporum. Most research
TABLE 4-13 Classes of Mycotoxins Produced by Fusarium oxysporum
|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 Fusarium 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 determine 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 associated 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 (Ncayiyana
1986; Hendricks 1999; Gelineau-van Wase et al. 2009). A strain of F. oxysporum 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 infections in immunocompetent people and 330 cases in immunocompromised people (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 immunocompetent 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 inoculum 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 infections lead to cosmetic disfigurement of affected nails, the infections are selflimited in immunocompetent hosts.
In immunocompromised hosts, fusariosis is a much more serious infection. Although many of the infections in this population begin as nail and superficial 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. Although increased Fusarium in the environment could lead to increased infections in immunocompromised populations, the population at risk in developing nations 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 potential 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.
An additional factor that should be considered in an evaluation of potential effects of Fusarium on humans is the paucity of effective antifungal drugs available 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 infection. Combination therapy with amphotericin B did not improve overall success. 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).
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 established, 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 numerous genotypes (Goddard et al. 2005). Recombination is accomplished by mating and meiosis but also could occur by nonsexual anastomosis and nonmeiotic recombination via the parasexual cycle (even though many fungi have mechanisms to limit nonsexual hyphal anastomosis to partners with almost identical genotypes). All those evolutionary processes apply to fungi used as mycoherbicides.
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 capabilities. Such mutations are probably due to the inactivation of one or more functional genes that are already in the strain’s genome. Under field conditions, mutations
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 recombination 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 appearance 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 concentrations of chlorate (Klittich and Leslie 1988). The mechanism underlying this mutagenic process has not been characterized, but it appears to have a multigenic base (Klittich et al 1988). In both those cases, mutations appear to be directed 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 selection should occur.
Mutations could affect mycotoxin production, provided that a mycoherbicide has the genes for toxin production. Mutations also could alter the synthesized toxin by blocking its biosynthetic pathway; this would lead to the accumulation of an intermediate in the pathway rather than of the end product. No on toxin production in the proposed F. oxysporum mycoherbicides are available.
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 exceptions.
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 reduce 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 effective 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 disease, 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-monthold 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 anecdotal observations suggest that the coca wilt disease persists and affects coca leaf production in some portions of the valley. In some areas, however, cultivation of coca has been moved to new fields to escape the disease (personal communication, 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 sporadic disease outbreaks.