Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops (2011) / Chapter Skim
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4 Fusarium oxysporum formae speciales as Candidate Biological Control Agents for Cannabis and Coca
4 Fusarium oxysporum formae speciales as Candidate Biological Control Agents for Cannabis and Coca
Pages 61-100
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oxysporum form species can have a wide host range and a single genotype is capable of parasitizing several different plants. For example, F
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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.
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oxysporum from severely diseased coca plants (Erythroxylum coca and E novogranatense)
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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)
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Assessment method Efficacy based on percentage of plants killed Field Studies Location Field experiments conducted in Vitulazio, Alvignano, and Portici, Italy, in 1974 Inoculum Air-dried straw-soybean inoculum used; applied by hand and mixed with top 10 cm of soil Application rate and Vitulazio field trial: 10 g/m2; inoculated plots seeded with C cultivars used sativa cultivars CS and SF Alvignano field trial: 10 g/m2; inoculated plots seeded with C
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sativa (cultivar not stated) Environmental conditions 28°C; 19-h photoperiod Assessment method Disease severity rating: 1 = healthy plant; 2 = wilting of lower leaves; 3 = wilting of 25-50% of leaves; 4 = over 50% of leaves wilted; 5 = dead plant Field Studies Location Kazakhstan field naturally infested with C
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, 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.
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(1997) performed three field trials in Hawaii with a ricealginate prill formulation of strain EN-4 of F
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deposited in and rate 2-cm-deep V-shaped trench and covered with 2-3 cm of soil Surface method: inoculum applied directly to the soil surface and covered with mesh screen to protect from predation Assessment method Plants in treated and nontreated soil evaluated for severity and mortality after 7, 8, 9, 10, and 15 mo Efficacy assessed with a disease-severity rating scale of 0-2: 0 = no disease, 1 = wilt, 2 = plant death of initial disease symptoms to plant death ranged from days to months. The average disease severity for all seven isolates tested was 1.1 (on a scale of 0-2)
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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
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From page 71... ...
(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.
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F oxysporum causes vascular wilt, as exhibited by leaf yellowing, loss of turgidity, necrosis, wilt, and plant death.
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. 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.
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oxysporum f.sp. erythroxyli inoculum for field trials.
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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.
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, 2 L distilled water plus 160 g of soybean meal, Per gram of fungus-colonized soil: alfalfa straw, or cottonseed meal or glycine-succinate-NaNO3 solution Barley straw plus cottonseed meal: 3.5 × 104 inoculated with the fungus, incubated for 3 wk in 2- to 3-L glass flasks Barley straw plus alfalfa: 2.7 × 104 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 Barely straw plus succinate-glycine-NaNO3 mixture: 2.5 × 104 determined after 5 wk Barley straw plus soybean meal: 1.46 × 104 McCain and Noviello 1985 F oxysporum f.sp.
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oxysporum f.sp. erythroxyli strain EN-4 produced by 3 × 107 CFU/mL produced with this method method of Hebbar et al.
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Bailey et al. 1997 33.6 kg/ha, inoculum composed of alginate prill formulation 5,336 34 erythroxyli -- coca containing 1 × 106 to 5.3 × 106 CFU F
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Fungus + barley straw + soybean meal; Air-dried inoculum produced on all straw-substrate mixtures and stored in plastic fungus + barley straw + alfalfa straw; bags at room temperature reportedly remained efficacious for 6 mo; seedlings fungus + barley straw + cottonseed meal; inoculated with formulations stored for 6 mo died within 18-20 d after inoculation; fungus + barley straw + glycine-succinate-NaNO3 solution; loss in efficacy noted for formulations stored for 9 mo and 12 m; seedlings inoculated all inoculated straw-substrate mixtures air-dried for 2 wk) with formulations stored for 9 mo and 12 mo died after 24 and 30 d after inoculation, respectively McCain and Noviello (1985)
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(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)
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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.
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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
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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
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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.
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. Infection of cannabis began to appear in field trials conducted in Kazakhstan in May and June, when temperatures were 2030°C.
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regime, in which populations fell below Treatments replicated three times detection levels after 17 wk Experiment repeated once a For all tests, alginate prill formulation (105 CFU/prill) was used and 10 prills were added to 25 g of soil.
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, <80% to Hatboro loamy sand >97% relative humidity, drip irrigation for first 10 d Red clay subsoil Hawaiian clay Experiments 1 and 2: fields previously grown with coca for 7 yr or more 29°C, 14-h daylight; 22°C, 12-h night for 7 wk Experiment 3: field not previously grown with coca Soils watered once per week, misted daily 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, 28, 56, and 229 d after treatment EN-4 distinguished from background Fusarium on Experiment 3: single samples taken 0 and 200 d after treatment basis of colony morphology and distinct orange color (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 Results EN-4 population was significantly greater than Experiment 1: 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 Population highest in top 7.6 cm of soil (maximum, 534 CFU/g 2 wk, declined to 1,060 CFU/g over next 5 wk after 1 mo, declined to 95 CFU/g over next 6 mo)
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1998 Formulation and application Rice alginate prill, wheat-flour kaolin, and rice and Rice alginate prill, wheat-flour kaolin, and rice and wheat-flour wheat-flour mixture mixture F oxysporum f.sp.
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, 20°C (air) , >97% humidity over first 10 d of treatment Soil sampling method Two samples taken 1, 4, and 6 wk after treatment Experiment 1: sampling method not specified Three samples taken 2 and 6 wk after treatment 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 treatment Samples taken from top 1 cm of soil and at 7.6-15.2 cm Results All formulations resulted in greater populations Experiment 1: No data on EN-4 populations reported of EN-4 in soil than in untreated controls; rice Experiment 2: alginate prill had lowest increase All three formulations resulted in greater populations of EN-4 in EN-4 populations highest in top 1 cm of soil top 1 cm of soil than in control (mean maximum, 15,135 (mean, 5,587 CFU/g)
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. Thus, the potential exists for the proposed mycoherbicides to move out of the geographic areas where they are applied.
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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.
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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.
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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.
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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)
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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)
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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.
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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.
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. Genetic variation can become established, or fixed, in fungal populations by natural selection or by chance.
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WHAT WE CAN LEARN FROM A NATURAL EPIDEMIC OF FUSARIUM OXYSPORUM F.SP. ERYTHROXYLI WILT OF COCA IN PERU The natural occurrence of diseases is common in plant populations, and plants that are intensively grown as crops are particularly vulnerable to periodic devastating epidemics.
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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?
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