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Will charged water ions sprayed onto plants keep insects off?

Will charged water ions sprayed onto plants keep insects off?



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I had stumbled on an article about a Dutch firm that devised an irrigation system that used mist of ionized water droplets that they claim keeps insects away from the plants. This break through would revolutionize eco-friendly organic agriculture by reducing the need for insecticides. They also claim nitrates are also formed by the process and embedded in the mist to supply nitrate fertilizers as well. Thus eliminating the need for extra chemical fertilizers.

I tried to find the site again and the company but not coming up on search. Are there any merits to these claims? How would such a process or device work?


Will charged water ions sprayed onto plants keep insects off? - Biology

The Killer Chemicals for Control of Agriculture Insect Pests : The Botanical Insecticides

Nuclear Institute for Agriculture & Biology (NIAB), Faisalabad, Punjab, Pakistan

This paper is likely to be fruitful by adding a share in the knowledge about use of botanicals and their processing into suitable manner as candidates for inclusion in the insect pests management with special focus on most destructive products. The use of synthetic insecticides for controlling of insect pests globally may have problems such as their persistent toxicity in food, the subsequent development of resistance in insect populations, effects on non-target organisms and other adverse environmental impacts. Of these, for sound management of pests there is an increasing interest in biotic control using plant products, which can prove eco-friendly with highly reduced negative effects on environment. Botanicals pesticides are plant derived materials such as from rotenone, pyrethrum, sabadilla, ryania, etc., used universally from commercial agriculture to institutions, homes and landscapes. Sometimes, nicotine products, although natural, yet are not considered bio-rational due to their high mammalian toxicity. The most potent bioactive compounds responsible for insecticide properties in b otanicals are alkaloids, non-proteic amino acids, steroids, phenols, flavonoids, glycosids, glucosinolates, quinones, tanins, terpenoids, salanine, meliantrol, azadiractin, piretrolone, cinerolone and jasmolone acting as contact poisons, ingestion or stomach poisons, feeding deterrences, repellents and confusants, leading to finally death of the insect victims. Some newer botanical insecticides have low mammalian toxicity and these include products made from extract of neem tree seeds, which is labeled for many vegetable crops. Azadirachtin is the active ingredient that works by inhibiting development of immature stages of many insects and by deterring feeding by adults. Garlic and hot pepper-based materials are other low-toxicity botanicals used by some growers, although their efficacy is somewhat certain. Botanical insecticides show significant fatal effects, antifeedant with significantly lower feeding rates and repellent effects against insects when their populations are low to moderate in size. Botanicals pesticides are biodegradable, barely leave residues in the soil and are less likely to harm humans or animals. In addition, these are cheaper and more accessible in less developed countries. Most botanicals are broad spectrum, so along with pest control they may kill beneficial insects too. However, these tend to be moderately toxic to peoples and wildlife, and many are irritating to mucous membranes. Use of powdered and extract forms of test plants is rec ommended for small scale farmers of developing countries due to the simplicity of application, easy removal and non-toxic effects even if consumed by humans as having medicinal properties.

Botanical Pesticide, Plant Derived Material, Insecticide, Knockdown, Mortality

Received: July 26, 2015
Accepted: August 6, 2015
Published online: August 13, 2015

@ 2015 The Authors. Published by American Institute of Science. This Open Access article is under the CC BY-NC license. http://creativecommons.org/licenses/by-nc/4.0/

C onventional broad-spectrum insecticides such as carbamate and organophosphate compounds can provide good protection of crops and grains from feeding by insects and other pests. These neurotoxins have activity through contact and ingestion, and cause rapid knockdown and death in the target insects. However, use of plant based pesticides, either in crude form or by processing into different formulations, is one of the many possible avenues explored with regard to biological control (Isaacs et al., 2004). Certain plant families, particularly plant products of Rutaceae and Myrtaceae have shown, in previous observations, repellent, insecticidal, anti-feedant, and growth regulatory properties against insect pests. Most of the plants thrive in rough environmental conditions so they have developed a multitude of defense mechanisms against natural enemies in the course of evolution. Among these are morphological and subtle chemical defense mechanisms against insects and other pests that do not generally cause immediate death but interfere with their vital biochemical and physiological functions (Neoliya et al., 2007 Yankanchi and Gadache, 2010).

Members of family of aromatic plants such as Eucalyptus species are usually highly aromatic and have monoterpenoids as bioactive principles with insecticidal properties. Due to their volatile and lipophilic nature, monoterpenoids can rapidly penetrate into insect’s cuticle and cause their mortality by interfering with their vital physiological functions (Isman, 2000). It is possible to create effective and natural insecticides from these substances to protect crops to cope with pests that unlike to wild plants may have lost their capability through cultivation. Botanical pesticides have many advantages over synthetic ones and may be more cost-effective as a whole, considering the environmental cost of chemical alternatives. Most botanicals are broad spectrum, so along with pest control they may kill beneficial insects, too. Many plant products could be expected to break down naturally outdoors and not cause any long-term toxic effects. Botanicals are generally short-lived in the environment, as these are broken down rapidly in the presence of light and air, thus they do not provide pest control for very long time or perhaps several days (Ahmad et al., 2011 Sarwar, 2012 a).

The botanicals insecticide can work by ingestion, through contact, as a deterrent and by disrupting developmental processes. Ingestion is when the moth larvae consume the pesticide and are poisoned. Contact poisoning is when the solution kills the moth larvae through their skin or other tissue. A deterrent is when the insecticide prevents the moth larvae from feeding and they starve. Finally, certain pesticides, notably oil from the neem tree, disrupt the hormones that control molting and other processes. Aphids, spider mites, and other pests can cause serious damage to flowers, fruits, and vegetables. These creatures attack garden and farm in swarms, literally draining the life from crops and often inviting disease in the process. Many chemical pesticides can prove unsafe for the environment or may make fruits and vegetables unsafe for consumption, however, there are many homemade, organic options to turn to war against pests (Sarwar, 2010 2013 a).

2. Considering Botanical Insecticides

For botanical insecticides, sometimes also referred to as ‘botanicals’, the earlier scientists have reported the development of inhibitory action and reproductive sterility effects against insects on agricultural crops that are under constant assault by pests, making use of insecticides essential to reduce losses. Synthetic insecticides such as organophosphates are important and effective tools in modern crop management. However, these pose serious threats to the environment and to peoples. Humans come in contact with dangerous pesticides on food, in water and in the air near farms. This ‘pesticide drift’ occurs when pesticide dust and spray travel by wind to places unexposed to pesticides. Almost ninety eight percent of sprayed pesticides do not reach to their target pests. They penetrate to groundwater, pollute streams and harm wildlife, including natural predators of the targeted pests. In short, global ecology is facing severe threat from the use of pesticides so the search for ecologically safe methods to control insect pests of crops and stored food products is an awe inspiring field of research (Sarwar and Sattar, 2012 Hina et al., 2015).

Botanical pesticides are naturally occurring insecticides derived from plants that have been formulated specifically for their ability to control insects. Botanical insecticides degrade readily in the sunlight, air, and moisture, breaking down into less toxic or nontoxic compounds and posing less risk to non-target organisms. But this requires precisely time of application and may also need to make more frequent applications if someone observes additional damage. Botanicals may not kill an insect for hours or days, but they act very quickly to stop its feeding. It must be noted that data on effectiveness and long-term toxicity are unavailable for certain botanicals. Botanicals tend to be less expensive than synthetic pesticides, and some are not as widely available. Also, the potency of some botanicals may differ from one source or batch to the next. Most botanicals do not damage plants and many botanicals are low to moderate in toxicity to mammals, but there are exceptions. For example, both inhalation and skin exposure to nicotine preparations can cause death. Also, rotenone is similar in toxicity to the common synthetic insecticides carbaryl and diazinon (Rajput et al., 2003 Khan et al., 2010). Plants produce numerous chemicals, many of which have medicinal and pesticidal properties. More than 2000 plant species have been known to produce chemical factors and metabolites having value in pest control programs. Many plant species produce substances that protect them by killing or repelling the insects that feed on them. For example, the Douglas fir has a special sap that wards off beetles if it is attacked. Neem trees produce oil that alters the hormones of bugs so that they cannot fly, breed or eat (National Academy of Sciences 1992).

G arlic produces allicin, which gives to garlic its smell and healthful properties. Garlic does not contain allicin itself, but when the cloves are crushed, two chemicals inside react to form allicin. This is why garlic does not smell until anyone crushes it. Allicin has been shown to have antifungal, antibiotic and antiviral properties, and researchers believe that it may help to prevent cancer. Garlic oil has been used as an insect repellent, and may be toxic to certain insect eggs. It is possible that in high concentrations, the antibiotic effects of garlic become lethal to the moth larvae. Garlic-based insecticide is highly concentrated and somewhat slower to cause 50% mortality but it has the second highest eventual lethality. It may have acted as a repellent to the worms, making them not to eat their food, but it may also have contact-based effects. Green chilies and other hot peppers contain a natural substance called capsaicin that creates the hot, spicy effect. Capsaicin at 10 parts per million causes a persistent burning sensation. The intense flavor comes from the large hydrocarbon ‘tail’ of the molecule. Capsaicin works by opening doors in the cell membranes that enable calcium ions to flood into the cell, making it trigger a pain signal that is transmitted to the next cell, and onward and so on. Extremely high concentrations of capsaicin are toxic and destroy cells by stopping the production of certain neurotransmitters that enable cellular communication. By boiling the chilies, it is possible to isolate and concentrate the capsaicin and other chemicals. Because greater wax moth larvae are small, soft-bodied insects, sufficiently high concentrations might contain enough capsaicin to destroy cells and kill them. Another possibility is the acidity of hot peppers and the soft skin of a wax moth might be damaged by the pepper's chemicals (Liener, 1986). Limonoids, are extremely bitter chemicals present in citrus seeds, act as antifeedants or antagonize ecdysone action in many species of coleopteran (Schultz, 1994). A variety of these plants contain secondary metabolites that show insecticidal activity against several coleopteran and dipterans species (Salvatore et al., 2004).

Botanicals are basically secondary metabolites that serve as a means of defense mechanism of the plants to withstand the continuous selection pressure from herbivore predators and other environmental factors. Several groups of phytochemicals such as alkaloids, steroids, terpenoids, essential oils, and phenolics from different plants have been reported previously for their insecticidal activities (Canyonb et al., 2005). Research studies have been carried out to evaluate insecticidal action of two plant products on a major stored-product insect Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). The plant species studied included, Psidium guajava (L.) (Guava, leaves: Myrtaceae) and Citrus reticulata (Kinnow, peel and leaves: Rutaceae). Two formulations viz., powder and ethanol extract of each plant have been prepared. Repellency has been tested using the filter paper test whereas mortality, weight loss protection and anti-feedant potential of all treatments evaluated by using whole wheat grains. The results reported that all tested treatments have significant effects pertaining to all variables analyzed and ethanol extract has been found to be remarkably more potent than powder form of same plant. Furthermore, leaves and peel of C. reticulata do not differ significantly pertaining to their toxicity against adult T. castaneum but proved stronger than P. guajava (Iram et al., 2013).

Common botanical insecticides include pyrethrum or pyrethrin that is a dust or extract derived from the pyrethrum daisy. These products are registered for use on animals to control fleas, flies and mosquitoes. They are also used as indoor household sprays, aerosols and ‘bombs’. Pyrethrins are sometimes combined with rotenone and ryania or copper for general use in gardens. Sabadilla is effective against certain true bug insects such as harlequin bugs and squash bugs, which are difficult to control with most other insecticides. Sabadilla is highly toxic to honey bees, so avoid spraying it when bees are present. Ryania may be used on citrus, corn, walnuts, apples and pears for the control of such pests as citrus thrips, corn borer and codling moth. Nicotine is used in greenhouses and gardens to control soft-bodied sucking pests such as aphids, thrips and mites. Because it can be toxic to humans, nicotine teas are not recommended as a way to control garden or household pests. Limonene and linalool are especially common in flea dips and pet shampoos. And the compound azadirachtin, derived from the neem tree, is sold under various names, and it may be used on several food crops and ornamental plants to control white flies, thrips, mealybugs, and other pests (Sarwar et al., 2005: 2012 2013). The most prominent active constituents from plants which are responsible for the b otanical insecticides properties are alkaloids, non-proteic amino acids, steroids, phenols, flavonoids, glycosids, glucosinolates, quinones, tanins, terpenoids, salanine, meliantrol, azadiractin, piretrolone, cinerolone and jasmolone. Mode of action for plant derivatives used for insect pests management is as contact poisons, ingestion or stomach poisons, feeding deterrences, repellents and confusants, which paralyze nerve activity, respiratory arrest, and act on the central and peripheral nervous system leading to convulsions and finally death of the insect victims (Rahuman et al., 2008 Silva-Aguayo, 2013).

Neem (Azadirachta indica) is a fast-growing broad-leaved evergreen tree that has many uses for centuries and one of those is as insecticide action. It can work in several ways, as a sterilant, a deterrent, an anti-feedant and as an insect growth regulator. However, it is relatively recent as a commercially available product and its effectiveness is not yet at the level of the other botanical discussed herein. It works best when insect populations are low to moderate in size. The product needs to be applied as soon as the pest appears and then re-applied, as often as every week, as long as the pest is active. This may become time-consuming (at the commercial level) and not necessarily cost effective. It is expected that more effective neem products can appear eventually. The A. indica tree contains an unusually high number of potent compounds, notably the chemical azadirachtin has strong resistance to termites that makes it a useful construction material. Azadirachtin harvested from the seeds, commonly offers broad range insect control and it is known not highly toxic to mammals. The bright red adult lily leaf beetle (Liliocerislilii) and its larvae are voracious feeders of all true lilies and it is a devastating pest. Neem has been effective against this pest when utilized as described in the above line.

Nicotine sulfate is toxic to most warm-blooded mammals and requires extreme caution when handling and applying. It kills insects by disrupting their nervous systems, causing vital functions to cease and finally the death. The tobacco is steeped in water to manufacture it, and it is then sprayed onto plants and sometimes livestock to ward off insects.

3.3. Pyrethrum or Pyrethrins

Pyrethrum or Pyrethrins are widely-used botanical insecticides made from the chrysanthemum plant. Pyrethrins can incapacitate most insects immediately however, many insects can recover from its effects unless pyrethrins are combined with other insecticides.

Rotenone insecticides are made from the roots of the Leguminoceae or Fabaceae plant family like from roots of Derris plant species. Rotenone is an odourless and colorless, crystalline ketonic chemical compound used as a broad-spectrum insecticide and pesticide. Rotenone insecticides are mildly toxic to humans and many animals, fish in particular. It stops insects from feeding, leading to their demise via starvation and interference with respiration at the cellular level.

It comes from the stems and roots of the shrub Ryaniaspeciosa. It acts as a stomach poison and keeps insects from feeding leading to death. The Ryania is moderately toxic to mammals, if ingested.

The botanical pesticide manufactured from the seeds of sabadilla lily, is possibly the least toxic of all organic insecticides. It is sold as a powder and is a contact poison, but unfortunately due to its low toxicity, sabadilla may not stop density of all insects. It is effective against leafhoppers, stink bugs, thrips, most caterpillars and squash bugs. It repels slugs, snails and many crawling pests. It is toxic to honeybees and degrades rapidly on exposure to sunlight and leaves very minimal trace toxicity.

4. Preparing of Botanical Pesticide

All of botanical pesticides are made from common plants that grow in many parts of the world and can be purchased quite cheaply. It is easy for anyone to produce them in kitchen and method is same that is already used with other plants such as neem tree leaves, and could be expanded to industrial capacities.

i. To prepare botanical pesticide, cut off 200 g of plant parts to be tested, wash thoroughly, chop them up in a food processor and add one liter of distilled water. It is important that there is nothing in the water that might affect the pesticide and then concentrate it by boiling to extract the substances from the plant parts, and strain out the sediment.

ii. The same could also try with 400 g of peeled garlic cloves and 250 g of green chilies by weighing out a cup of each, and calculate the concentration of plant substances per liter of solution. All the vegetables should be washed and chopped prior to use.

iii. Blend the vegetables together in an electric blender, and a thick, chunky paste should form.

iv. Add the vegetable paste to 2 cups (500 milliliters) of warm water and thoroughly mix the ingredients together.

v. For neem seed extract, powder obtained by crushing 400 g of seeds is dissolved in 2 litres of water and stirring well. Then allow the solution to soak for about 12 hrs. The resulting solution is filtered through a thin cotton cloth, and diluted with water to bring the solution to 10:1, for the control of pest attack.

vi. Pour the solution into a plastic or glass container and allow it to sit for 24 hours, if possible keep it in a sunny location, or if not, at least keep the mixture in a warm spot.

vii. Strain the mixture, pour the solution through a strainer removing the vegetables and collecting the vegetable-infused water into another container that is the desired pesticide.

viii. Pour the pesticide into a squirt bottle and make sure that the spray bottle has first been cleaned with warm water and soap to rid it of any potential contaminants.

ix. Spray the plants with pesticide and treat the infected plants every four to five days. After three or four treatments, the pests may scatter. If the area is thoroughly covered, this pesticide should keep bugs away for the rest of the season.

x. The solutions prepared here should be mixed well with soap solution at the rate of 10 g/ 1 litre of extract before spraying to facilitate uniform spread of the neem solution. It is recommended that the application or spraying of the all botanical pesticide should be carried out only in the late afternoon of the day.

5. Testing Procedure of Botanical Pesticide

Botanical insecticides can be tested each at three concentrations, on populations of adults or larvae of insect. The medium and lowest concentrations are diluted 3:1 and 5:1 from the fully concentrated solution. After creating test insect populations, allow the adults or larvae to get time used to their environment and eliminate natural deaths, then spray the known numbers with natural insecticide and thereafter track their survival rates. The highest concentrations of all of the insecticides can cause mortality within one to three days of spraying. Every day try to track the numbers of dead and live adults or larvae of insect population for each concentration of the insecticide. It can be analyzed, 1) the mortality rate (how many died on each day), 2) how many days are needed to achieve lethality to 50% of the population, or LD 50, which is the standard used when testing products, and 3) total mortality after 15 days.

6. Switch Over to Integrated Pest Management (IPM)

In order to minimize the use of hazardous chemical pesticides and to manage the insects, pests and diseases attack as well as to increase the crop productivity, it is necessary to implement a scheme to strengthen and modernization of pest management approach by adopting Integrated Pest Management (IPM) as cardinal principle and main plank of plant protection strategy in overall crop production program. Under this program, it should have a mandate to popularize adoption of integrated pest management through training and demonstration in crops inter-alia promotion of biological control approaches in plant protection technology. Settlement of the laboratory to land gap can be narrowed down through proper training and education to the farmers by way of expert and regular extension services. The managed pest management system eliminates or mitigates economic and health damage caused by pests minimizes the use of pesticides, and the risk to human health and the environment associated with pesticide applications and uses integrated methods, site or pest inspections, pest population monitoring, an evaluation of the need for pest control and one or more pest control methods including sanitation, structural repairs, mechanical and biological controls, other non-chemical methods, and if nontoxic options are unreasonable and have been exhausted, use least toxic pesticides (Sarwar, 2012 b 2013 b 2013 c).

In the light of the findings of present study, it could be stated that botanicals pose reasonable insecticidal properties against insects and have shown promising effects for plants and seeds protection, so these might be used in pests population managing in crops and stored grains. Other potential experiments could include trying of different concentrations and varying the spraying frequency of the botanicals, as well as spraying the larvae and their food separately to see if the botanical is effective through ingestion or as a deterrent, as opposed to killing by contact. It could be tried different species of pests to see which botanicals are most broadly effective. There could also try to test different parts or ingredients of the plants, or mixing of solutions together to create more potent botanicals. It could be tested other botanicals to see if these have similar effects or could also try testing relatives of botanicals such as different types of plant families and such experiment could lead to derive many more pesticides that could improve the farming processes used today. Members of family of aromatic plants like Eucalyptus, and members of the families such as Solanaceae, Asteraceae, Cladophoraceae, Labiatae, Miliaceae, Oocystaceae and Rutaceae are available for maximum months in a year. So, use of their body parts (leaves, stems, barks, flowers, fruits, roots, seeds) as alternative to conventional insecticides is recommended due to their relative abundance and accessibility throughout the year. Further investigation for the isolation of individual components of plant prats to determine the exact mode of action of the active components, and their effect on target and non-target organisms is advocated for their possible incorporation in botanical insecticides on commercial scale.


Sweden: Plant Research Injection without Damage Allows New Insights into Plant Physiology

Eleni Stavrinidou and her research group at the Laboratory of Organic Electronics, Linköping University, have used bioelectronics to influence transpiration in a tobacco plant, without harming the plant in any way. This is an important success, since plants do not repair damaged tissue as animals and humans do.

The Ion pump consists of an electrode made from a conducting polymer inside a tiny container connected to a channel based on a polymer electrolyte.

Linköping/Sweden — Research in the Electronic Plants group at the Laboratory of Organic Electronics, Campus Norrköping, follows two main avenues. In one, scientists incorporate electronic circuits into plants, such as roses, in a method of storing energy. In the other, they are seeking ways to influence plant functions with bioelectronic devices aiming, for example, to give plants greater resistance to environmental stress.

The scientists can implant electronic devices into plants without damaging the plant. This research field is new, but they are starting to be able to influence plant physiology. Eleni Stavrinidou leads the research group in Electronic Plants at the Laboratory of Organic Electronics, Linköping University. She hopes that this will become an effective tool to study how plants function, or find applications in agriculture and forestry.

Germany: Biodegradable Crop Protection

Common Stress Hormone

Scientists Iwona Bernacka-Wojcik and Miriam Huerta have managed to electronically deliver a common stress hormone known as ABA into a tobacco plant. The plant normally secretes this hormone when subjected to stress, for example, during drought or other extreme weather conditions. A solution containing the hormone is also sometimes sprayed onto plants in shops, to keep them fresh longer.

The researchers showed that after the hormone was delivered it spread through the leaf tissue, and that the small pores, the stomata, on the leaf surface closed, to prevent the release of water. The plant must optimize the opening and closing of the pores because when they are open it carries out photosynthesis, but at the same time it loses water. Using the new generation of ion pump, with a capillary form whose diameter is no thicker than a hair, they could electronically deliver ABA molecules into the leaves of tobacco plants without harming the plant. If the moisture remains in the plant, it would become more resistant to, for example, drought, says Eleni Stavrinidou.

The lack of any damage to the leaf and the plant is an important part of the success, since plants do not repair damaged tissue as animals and humans do. Instead, the plant discards the damaged leaf or branch, and replaces it, in the optimal case, with a new leaf or shoot.

The Ion Pump 2.0

The tiny ion pump was developed a couple of years ago at the Laboratory of Organic Electronics. It consists of an electrode made from a conducting polymer inside a tiny container connected to a channel based on a polymer electrolyte. Ions are led through the thin channel out to exactly the correct position — inside a root fibre or leaf vein of a plant. The container in this case is filled with ABA. When a voltage is applied across the electrodes, one in the container and one external, the charged substance is transported out of the channel into the tissue. The rate of delivery of the substance is directly proportional to the current. Only the active substance is pumped out, nothing else, and there is no return flow to the pump. The scientists can give the plant the exact dose it needs, with high precision.

The results from the research — which may also contribute to a deeper understanding of plant physiology — have been published in the journal Small, Advanced Science News.

References: Implantable Organic Electronic Ion Pump Enables ABA Hormone Delivery for Control of Stomata in an Intact Tobacco Plant, Iwona Bernacka-Wojcik, Miriam Huerta, Klas Tybrandt, Michal Karady, Mohammad Yusuf Mulla, David J. Poxson, Erik O. Gabrielsson, Karin Ljung, Daniel T. Simon, Magnus Berggren, and Eleni Stavrinidou, Small 2019. DOI 10.1002/smll.201902189


APPLICATION OF NANOPARTICLE-MEDIATED GENE TRANSFORMATION METHODS IN PLANT GENETIC ENGINEERING

Magnetofection

Magnetic nanoparticle-based transformation (magnetofection) was used for drug delivery in the 1970s and for the transfection of mouse cells in the year 2000 (Dobson, 2006 ). This method provides a useful way to transfer genes into the cell nucleus by the application of a magnetic field (Scherer et al., 2002 ). Nanoparticles are classed according to their structure, size and shape. There are three types of magnetic nanoparticles: pure metals, metal oxides and magnetic nanocomposites (Cardoso et al., 2018 ). Notably, the magnetic nanoparticle Fe3O4 and Fe2O3 are the ideal materials for a variety of field applications and for fundamental investigations because of their large surface area, small size, low sedimentation rates, high thermal stability, stability and low toxicity (Cardoso et al., 2018 ).

Recent studies have indicated that magnetic nanoparticles have been applied mainly in animal science and medical research (Rozita, 2020 ), but there has been one successful study of magnetofection in plants. Zhao et al. ( 2017 ) developed a simple and fast method for producing transgenic seeds without tissue culture regeneration mediated by magnetic nanoparticles. Magnetic nanoparticles were packaged with plasmid DNA and forced into pollen by a magnetic field. The plasmid DNA–magnetic nanoparticle–pollen complex entered the plant through the typical pollination process and was integrated into the genome of the offspring. They chose Gossypium hirsutum (cotton) as a representative plant species, and the resulting transgenic cotton generated seeds with a stable inheritance of the transformed DNA (Figure 2).

Although the magnetofection method avoids conventional transformation and regeneration, it still has some disadvantages. The pollen apertures of some species may not be suitable for magnetic nanoparticle-based transformation. In addition, the magnetofection method is not suitable for maternally inherited organelles, such as chloroplasts and mitochondria (Ruf and Bock, 2017 ). Nonetheless, magnetofection may open new avenues for many species without transformation and regeneration methods.

Carbon nanotubes

Carbon nanotubes (CNTs) are typically classified into two main categories: single-walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs). SWCNTs consist of a graphene layer that is rolled into a cylindrical nanostructure with a diameter of 0.7–3.0 nm, and MWCNTs are formed by multiple SWCNTs and have a diameter of 220 nm (Hendler-Neumark and Bisker, 2019 Mohanta et al., 2019 ).

The SWCNTs serve as gene-delivery vehicles that can penetrate the membranes of organelles and have many advantages in plant genetic engineering (Kwak et al., 2019 ), such as biocompatibility, high aspect ratio, high surface area to volume ratio and exceptional tensile strength (Hendler-Neumark and Bisker, 2019 Mohanta et al., 2019 ). As a result of their high surface area to volume ratio, SWCNTs can be loaded with large quantities of DNA for transfer into plant cells (Burlaka et al., 2015 Kwak et al., 2019 Demirer et al., 2019a , 2019b , 2019c ). Furthermore, SWCNTs protect DNA from degradation in mammalian systems. SWCNTs may also deliver small interfering RNA (siRNA) and plasmid DNA into model and non-model plant cells for gene silencing (Demirer et al., 2019a , 2019b , 2019c ). Finally, the cargo–nanoparticle complexes formed by SWCNTs and their loaded cargoes have strong intrinsic near-infrared (nIR) fluorescence, allowing them to be tracked in real time (Li et al., 2014 Hendler-Neumark and Bisker, 2019 ).

The application of carbon nanotubes in plants has been limited because of the cell wall. To investigate the cell penetration ability of MWCNTs, Catharanthus roseus protoplasts were used. The results showed that MWCNTs can move through protoplast membranes, and short MWCNTs (<100 nm) target the plastids, vacuoles and nuclei (Serag et al., 2011 ) however, protoplast preparation is time consuming. Therefore, Serag et al. ( 2012 ) combined cup-stacked carbon nanotubes (CSCNTs) with cellulase, allowing them to traverse the cell wall. Intriguingly, SWCNTs can penetrate mesophyll cell walls and protoplasts in an energy-independent manner (Yuan et al., 2011 ), thus extending the application of this method in plant genetic engineering. Further research on CNTs in living plants, such as Eruca sativa, G. hirsutum, Nicotiana benthamiana and Triticum aestivum has shown that DNA–CNT conjugates not only infiltrate DNA into plant cells but also avoid polynucleotide degradation (Demirer et al., 2019a , 2019b , 2019c ).

After this breakthrough in the delivery of DNA into plant cells by CNTs, researchers have focused their attention on verifying different cargoes and gene functions. In the RNA interference (RNAi) process, siRNA plays an important role in degrading mRNA molecules. Agrobacterium-mediated transformation is an effective method for delivering siRNA, but is limited by plant species therefore, a new method is needed to address this problem. An SWCNT-mediated siRNA delivery approach has emerged with high efficiency, high throughput and high gene silencing efficiency at the mRNA level (Demirer et al., 2019a , 2019b , 2019c ). The delivery is passive and can be tracked by autofluorescence, and the siRNA can silence several genes at the same time.

Conventional plant genetic engineering methods (Agrobacterium-mediated transformation or gene-gun transformation) mainly deliver DNA into the nucleus, where it integrates into the genome, leading to the migration of genes out of transgenic plants and resulting in enhanced herbicide resistance in weeds. Genetic engineering of the chloroplasts and mitochondria avoids this limitation by being maternally inherited. Thus, Kwak (Kwak et al., 2019 ) developed chitosan-complexed SWCNTs that specifically deliver DNA to the chloroplasts of different plants, such asE. sativa (arugula), Nasturtium officinale (watercress), Nicotiana tabacum (tobacco) and Spinacia oleracea (spinach). The aforementioned studies of CNT methods demonstrated high efficiency, no measurable toxicity and no transgene integration into the nuclear genome, and these studies can be performed without mechanical aid and are appropriate for model and non-model plant species. There are some challenges in the use of CNTs, however. The physical and chemical characteristics of CNTs, such as their low water dispersibility, entangled bundle formation and inert nature, may limit the scope of their use in plants. Moreover, the mechanisms of the interactions between CNTs and plants are unclear. In addition, the potential environmental toxicity of CNTs is not fully understood.

DNA nanostructures

Recently, nanoparticle-based DNA/RNA delivery systems have been widely used for gene delivery, drug delivery and the delivery of other therapeutic agents (Gao et al., 2020b Ouyang et al., 2020 Ryu et al., 2020 ). In contrast to other nanoparticle carriers, nanostructures composed of DNA are the basic elements of genetic material in plants, animals and microorganisms they are therefore easy to metabolize, are highly efficient and are non-toxic(Li et al., 2019 ). In addition, DNA nanostructures have many merits. They have excellent biological stability and avoid biodegradation by nucleases (Kim et al., 2020 ). At the same time, they show high biocompatibility with the target tissues and do not trigger a protective immune response (Zhan et al., 2020 ).

Furthermore, the DNA nanostructure technique is modular, flexible and easy to perform in plants (Zhan et al., 2020 ). Currently, several DNA nanostructure patterns are used, such as nanostrings, nanocubes, nanostars and 3D geometric figures (e.g. icosahedrons and tetrahedrons). The cargoes (DNA/RNA) can be placed inside or outside the DNA nanoparticles and the nanoparticle-based DNA/RNA delivery systems can transport the cargoes across the lipid bilayer of the cell membrane, allowing it to reach the nucleus and perform its function. The cell walls in plant cells may form a natural obstacle to the delivery of biological molecules such as DNA and RNA, however. Thus, Zhang et al. ( 2019 ) developed DNA nanoparticles as a platform for siRNA delivery in plants. The efficiency of siRNA gene silencing was determined by the size, shape, stiffness, compactness and attachment site (siRNA attachment locus) of the designed DNA nanostructures. The researchers evaluated the internalization of these nanostructures into plant cells by detecting the intensity of a fluorophore that was conjugated to the DNA strands at the attachment loci of the DNA nanostructures by hybridization. These DNA nanoparticles introduced siRNA into the leaf cells of E. sativa, Nasturtium officinale, Nicotiana benthamiana and Nicotiana tabacum by infiltration, and the researchers found that the efficiency of gene silencing was directly proportional to the nanoparticle internalization efficiency and the siRNA attachment locus. The DNA nanoparticle method is a promising method that can be applied to a wider range of plant species.

Peptide nanoparticles

To date, many cell-penetrating peptides (of approximately 30 amino acids in length) have been employed as biotransmitters to translocate various types of molecules, such as DNA, proteins and chemicals (Ahmed, 2017 Bolhassani et al., 2017 ). There are many obvious advantages to using peptides for gene delivery in plant cells, including cell wall/membrane penetrating capability, extracellular stability and intracellular DNA release (Chuah and Numata, 2018 .

Initially, Rosenbluh et al. ( 2004 ) found that fluorescently labeled histones can penetrate the plasma membrane when incubated with Petunia protoplasts. This was the first time that peptides were shown to have the potential to deliver macromolecules into plant cells. Later, many studies demonstrated that peptides can also deliver DNA into epidermal cells, root tip cells, tomato root cells (Chang et al., 2005 Chang et al., 2007 Lu et al., 2010 ) and tobacco BY-2 cells (Eggenberger et al., 2011 ). These results indicated that peptides can be used as fast and efficient cell-penetrating agents to transport cargoes into the cytosol of walled plant cells, and thus peptides can be used as fast and effective cell-penetrating agents to carry cargoes across the cell membrane and into the cytoplasm, even in the presence of the plant cell wall.

A recent study indicated that organelle-targeting peptides can transport DNA into specific organelles of intact plants (Ng et al., 2016 ). Thagun et al. ( 2019 ) fused a cell-penetrating peptide and a chloroplast-targeting peptide with DNA to generate peptide–DNA complexes that could carry DNA across the cellular membrane and effectively deliver the DNA to plastids. This method can be used to alter plant plastids and transiently regulate gene expression without the need for stable plant transformation.

Although some progress has been made in the efficient delivery and release of DNA from peptide–DNA complexes (Chuah and Numata, 2018 ), many issues remain unclear and need to be addressed. The mechanism of peptide–DNA association and dissociation has not yet been resolved, and DNA release from the peptide–DNA complex is inefficient in cells, resulting in the low expression of foreign genes. In addition, peptide–DNA internalization and tracking mechanisms remain unknown and limit the design of efficient peptide gene delivery vectors.

Clay nanosheets

Recent studies have shown that plants can take up exogenous siRNAs, hairpin RNAs (hpRNAs) and double-stranded RNAs (dsRNAs), and can silence the gene expression of pathogenic viruses, fungi or insects in order to protect themselves (Kaldis et al., 2018 Dubrovina and Kiselev, 2019 ). Naked dsRNA can be sprayed on the leaves of a plant and is absorbed into the plant’s vascular system, thus triggering RNAi after the plant is infected with a pathogen (Gogoi et al., 2017 ) however, naked dsRNA is unstable and prone to degradation, and is easily washed off the leaves. The available studies show that a new type of nanomaterial, namely layered double hydroxide (LDH) clay nanosheets, is non-toxic, degradable and difficult to remove by washing. Notably, dsRNA can be loaded onto these clay nanosheets and released continuously. Plants can take up the dsRNA from the clay nanosheet–DNA complexes and silence the target viral DNA to protect themselves from infection (Figure 3). This innovative nanotechnology is an ecologically friendly way to improve plant characteristics and quality without the need for plant gene transfer.

Although this technique has been applied in some plants, many questions remain to be solved. First, it is not clear how plants perceive external RNA. Second, the mechanism of dsRNA uptake and transport remains unknown. Third, the mechanism of dsRNA interaction between plants and pathogenic microorganisms is also unknown. In addition, to improve RNAi efficiency, the optimal conditions for RNAi induction, such as nanomaterial, RNA length and RNA concentration, need further study.


Transgenics

18.3 Herbicide Resistance

Broad-spectrum herbicides are chemicals that kill virtually all plants. Consider the notion that if a plant were developed that were resistant to a broad-spectrum herbicide, then crops of this plant could be sprayed with the herbicide and all weeds and competing plants would die except for the plants of the desired crop. Glyphosate is the active ingredient in most broad-spectrum herbicides, including a product known as Roundup™. It has the structure shown in Figure 18.3 . Glyphosate acts to inhibit the activity of 5-enolpyruvylshikimate 3-phosphate synthase (EPSP synthase), which is involved in the biosynthesis of the aromatic amino acids and tetrahydrofolate, ubiquinone, and vitamin K. These particular pathways are not used in mammals, fish, birds, or insects, so EPSP synthase presents an attractive target for killing plants.

Figure 18.3 . The structure of glyphosate, the active ingredient of Roundup™.

With glyphosate serving as such an effective herbicide, engineering plants that are resistant to this agent is the goal of the above biotechnology for enhanced crop production. One approach to obtaining plants with glyphosate resistance is to have them make more EPSP synthase than an application of glyphosate could inhibit. This can be achieved by inserting additional copies of the EPSP synthase gene into the plant genome, along with multiple enhancers in front of the inserted genes. Another approach is to give the plants a gene coding for a slightly different EPSP synthase, one that is resistant to glyphosate. The result in this case was Roundup Ready plants, which were produced by Monsanto (who also produced Roundup). A specific example of a Roundup Ready plant is soybean, a plant that is resistant to glyphosate ( Figure 18.4 ).

Figure 18.4 . Roundup Ready™ soybeans.

Photo from flickr.com/photos/[email protected]/3810996291/in/set-72157622010267260/ .

Questions have arisen as to whether the produce of Roundup Ready plants is affected by exposure of the plants to glyphosate. One line of reasoning is that herbicides and pest control agents that are sprayed onto crops must gain FDA approval prior to use with the food supply. These agents have been deemed safe, so a plant that has been exposed to these agents is supposedly safe for consumption if it has been properly washed. A counterexample to this reasoning is DDT, which is a very effective insecticide that is still used in parts of the world to control mosquitoes to combat malaria. One problem with DDT is that it is not metabolized very quickly, so prolonged consumption of food that has been sprayed with DDT can lead to a buildup of the chemical in fat cells, eventually leading to toxic effects. The presence of DDT is not limited to plants, though, as it will be present in birds that may consume some of the crops and in fish due to runoff of rainwater into rivers and lakes. These multiple points of entry into the food chain led to DDT levels that eventually adversely affected humans, leading to premature births and low birth weights. This counterexample is a vivid illustration that a governmental agency is not infallible, despite its best testing efforts. However, this is only a singular counterexample and is not intended to cause one to doubt every single pesticide in use today. The debate whether to eat only organic produce or not cannot be resolved, since arguments against existing pesticides and herbicides are based upon future results that have not been realized.

Keep in mind that the only difference between organic foods and regular crops is whether or not pesticides or herbicides have been used during cultivation. A problem with organic crops is that they will, by necessity, cost more to produce. Without pesticides, there will be increased crop loss due to pests. The monetary amount of crop loss is expected to exceed the amount of money that would have been spent on pesticides. As a result, the price of the produce will be inherently higher if the grower is to break even or realize a profit.


Common stress hormone

Scientists Iwona Bernacka-Wojcik and Miriam Huerta have managed to electronically deliver a common stress hormone known as ABA into a tobacco plant. The plant normally Ion pump in plant Photo credit Thor Balkhed and Miriam Huerta/L secretes this hormone when subjected to stress, for example, during drought or other extreme weather conditions. A solution containing the hormone is also sometimes sprayed onto plants in shops, to keep them fresh longer.

The researchers showed that after the hormone was delivered it spread through the leaf tissue, and that the small pores, the stomata, on the leaf surface closed, to prevent the release of water. The plant must optimise the opening and closing of the pores because when they are open it carries out photosynthesis, but at the same time it loses water.

&ldquoUsing the new generation of ion pump, with a capillary form whose diameter is no thicker than a hair, we could electronically deliver ABA molecules into the leaves of tobacco plants without harming the plant. If the moisture remains in the plant, it becomes more resistant to, for example, drought&rdquo, says Eleni Stavrinidou.

The lack of any damage to the leaf and the plant is an important part of the success, since plants do not repair damaged tissue as animals and humans do. Instead, the plant discards the damaged leaf or branch, and replaces it, in the optimal case, with a new leaf or shoot.


J.L., F.M., and C.Y. conceived the original screening and research plants J.C. and Q.Z. performed the bioinformatics experiments Z.W. and J.P. provided technical assistance F.M. and C.Y. performed most of the experiments and data analysis J.L., F.M., C.Y., H.C, and Z.Q.F. wrote the article. All authors read and approved the manuscript.

Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.12922/suppinfo

Figure S1. bHLH transcription factors were differentially expressed upon M. oryzae infection in rice

(A) The differentially expressed transcription factors in response to M. oryzae invasion at 24 hpi in rice. The DEGs were acquired by applying q < 0.05 and two-fold as the cutoff. (B) Differentially expressed bHLH family genes in 2-week-old rice plants after infection with M. oryzae. Shown is hierarchical clustering of the 21 genes identified as differentially expressed in the pairwise comparison between Mock and 12, 24, and 48 hpi samples.

Figure S2. The OsbHLH6 expression level in OsbHLH6 overexpression, silencing, and mutant plants

(A–C) The OsbHLH6 expression levels in transgenic plants. Two-week-old rice seedling leaves were sampled for RT-qPCR assays. Values are means ± SD (n = 3 biological repeats). Two independent lines were used in each genotype. **Indicates significant differences from WT by student's t-test (P < 0.01). (D) Verification of the knockout lines by sequencing. OsbHLH6-Cas9-5 and -9 generated by CRISPR/Cas9 system showed single-nucleotide insertion in the OsbHLH6 target sequence, leading to the early OsbHLH6 translation termination.

Figure S3. Phylogenetic analysis and sequence alignment of OsbHLH6 and its homologs

(A) Sequence alignment of OsbHLH6, OsbHLH7, At4g29930, and At5g57150. Sequence alignments were performed by ClustalW (www.clustal.org). (B) Phylogenetic analysis of OsbHLH6 and its homologs from different plant species. The genes are from Oryza sativa (LOC), Zea mays (Zm), Brachypodium distachyon (BRADI), Triticum urartu (TRIUR), Glycine max (GLYMA), Solanum lycopersicum (Solyc), and Arabidopsis thaliana (At). The phylogenetic tree was generated using MEGA 7.0 (Kumar et al. 2016).

Figure S4. Sequence analysis of the OsbHLH6 promoter

The OsbHLH6 promoter was used to search the OsMYC2 binding motifs. G1, G2, and G3 indicate the three putative OsMYC2 binding sites. The black underlined sequence was used for the binding specificity assays. G3-m is fragment containing the sequence of C CACGCG (G3)G that is mutated to AAAAAAAA in the EMSA assay.

Figure S5. OsbHLH6 overexpression activates JA signaling and biosynthesis in rice

(A) OsbHLH6 acts in JA signaling. Two-week-old rice leaves were treated with 100 μM MeJA, and the leaves were sampled 8 hrs later. RT-qPCR was used to examine the OsJAR1 gene expression. ** Indicates significant differences from WT by student's t-test (P < 0.01). (B) and (C) The JA and JA-Ile content in OsbHLH6 overexpression and mutant lines. The two-week-old transgenic plant leaves were used to measure the JA and JA-Ile content. The experiment was repeated twice. Values are means ± SD (n = 5 biological repeats). * and ** indicate the significant differences from WT by student's t-test at P < 0.05 and P < 0.01, respectively.

Figure S6. Overexpression of OsbHLH6 leads to senescence-related gene expression

(A–C) The senescence-related gene expression in WT and OsbHLH6-OE plants. Two-week-old rice seedlings were used to evaluate the gene expression levels. RCCR1 (Os10g0389200) Osh36 (Os05g0475400) SGR (Os09g0532000). The experiments were repeated three times with similar results. D-3 and D-4 are two independent T1 OsbHLH6 overexpression lines. ** indicates the differences from WT by student's t-test at P < 0.01. Values are means ± SD (n = 3 biological replicates).

Figure S7. α-OsbHLH6 antibody specifically recognizes OsbHLH6 in rice

(A) Crude OsbHLH6 protein extract was probed by anti-OsbHLH6 antibody. Two-week-old Dex:OsbHLH6 and Cas9-9 mutant rice leaves were treated with or without 10 μM Dex for 6 h. The plant leaves were sampled and the crude protein extract was subjected to immunoblotting assay. Anti-OsbHLH6 antibody was used to probe OsbHLH6 protein. ACTIN was used to indicate the equal loading of the total proteins. (B) The OsbHLH6 was detected in rice nucleus. The rice leaves in (A) were used to purify nucleus. The OsbHLH6 protein in the nucleus was probed by the anti-OsbHLH6 antibody after separated by the SDS-PAGE gel. Anti-Histone3 (H3) antibody was used to probe the histone3 to indicate the equal loading for the immunoblotting.

Figure S8. OsbHLH6 NES reduces OsbHLH6 in nucleus

(A) YFP-OsbHLH6 NES showing the cytosolic localization in N. benthamiana. DAPI staining showing the nucleus. The confocal microscope was used to observe the fluorescence signals. The experiment was repeated at least three times. Bar = 50 μm. (B) BiFC analysis shows OsbHLH6 NES and OsbHLH6 form dimers in N. benthamiana cytoplasm. Unfused cYFP was used as a negative control. Bar = 50 μm. (C) OsbHLH6 NES reduces the nuclear localization of OsbHLH6. YFP-OsbHLH6 and cLUC-OsbHLH6 NES were transiently co-expressed in N. benthamiana leaves. Co-expressed YFP-OsbHLH6 and cLUC-EV were used as the negative control. Confocal microscope was used to observe the YFP-OsbHLH6 localization. Right panel showing the fluorescence intensity of YFP-OsbHLH6 in nucleus crossing the red bars. Bar = 50 μm. (D) The YFP-OsbHLH6 proteins in (C). The protein expression was examined by immunoblotting assays, showing the similar expression levels. The anti-GFP antibody was used to probe the YFP-OsbHLH6.

Figure S9. SA attenuates the interaction of OsbHLH6 and OsNPR1

(A) BiFC analysis shows the interaction of OsbHLH6 and OsNPR1 in N. benthamiana. Nucleus of leaf epidermal cells were stained with DAPI. Merged image shows that the fluorescence is not in the nucleus. Unfused cYFP was used as a negative control. Bar = 50 μm. (B) SA attenuates the interaction of OsbHLH6 and OsNPR1. The proteins were transiently expressed in N. benthamiana leaves. After 24 h of the transformation, 100 μM SA was sprayed onto the leaves every 6 h. Co-immunoprecipitation assays were used to assess the protein interaction. (C) YFP-OsbHLH6 intensity of indicated position in (B). ** indicates significant differences from mock at P < 0.01, by student's t-test. Values are means ± SD (n = 3 biological repeats). (D) BiFC analysis shows SA attenuates the interaction of OsbHLH6 and OsNPR1 in rice protoplasts. The plasmids carrying OsbHLH6-nYFP and OsNPR1-cYFP constructs were co-transformed into rice protoplast. 16 h after transformation, rice protoplasts were treated with 200 μM SA for 6 h. Lower panel shows the protein levels in the samples. The experiment was repeated at least three times. Bar = 5 μm. (E) BiFC analysis shows SA attenuates the interaction of OsbHLH6 and OsNPR1 in N. benthamiana. 36-40 h after transformation, the leaves were treated with 200 μM SA for 6 h. The experiment was repeated at least three times. Bar = 50 μm. Others are same as in (D). (F) OsNPR1 2CA interacts weakly with OsbHLH6 by Split luciferase assays. The OsbHLH6 was fused with cLUC fragments, and OsNPR1 and OsNPR1 2CA were fused to the nLUC fragments. cLUC-EV and GUS-nLUC serve as controls. The proteins were co-expressed in N. benthamiana leaves.

Figure S10. OsNPR1 sequesters OsbHLH6 in cytoplasm

(A) OsNPR1 induces OsbHLH6 export from nucleus to the cytoplasm. YFP-OsbHLH6 and OsNPR1-T7 were transiently expressed in N. benthamiana leaves. 200 nM LMB was injected to the leaves 12 h before the observation. Confocal microscope was used to observe the YFP-OsbHLH6 localization. Bar = 50 μm. (B) YFP-OsbHLH6 intensity of indicated position in (A) by red bar. (C) The immunoblotting shows the protein levels of YFP-OsbHLH6, GUS-T7, and OsNPR1-T7 in (A). The expressed proteins were probed with respective antibodies to show the equal levels in N. benthamiana leaves. ACTIN served as the internal reference. (D) OsNPR1 did not affect the subcellular localization of OsEIL1 in rice protoplasts. GUS-FLAG, OsNPR1-FLAG, and OsEIL1-GFP were transiently expressed in rice protoplasts. GUS-FLAG was used as a negative control for OsNPR1-FLAG. Bar = 5 μm. (E) The immunoblotting shows the protein levels of GUS-FLAG, OsNPR1-FLAG, and OsEIL1-GFP in (D). The expressed proteins were probed with respective antibodies to show the equal levels. ACTIN served as the internal reference.

Figure S11. SA and JA signaling pathways are spatiotemporally regulated during M. oryzae infection

(A) Expression of JA and SA responsive genes in rice plants. Two-week-old WT seedling leaves were inoculated for transcriptome assays. The z-scores of RPKM expression values are shown. **** indicates significant differences from WT at P < 0.0001. Each experiment was performed with two biological replicates. (B) The expression of the SA responsive genes, OsNPR1 and OsWRKY45, in rice seedlings after M. oryzae infection. RT-qPCR was used to evaluate the gene expression. Values are means ± SD (n = 3 biological replicates). * and ** indicate the significant differences from WT by student's t-test at P < 0.05 and P < 0.01, respectively.

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What is it and how does it work?

When sprayed onto the leaves of any plant, SinC’s homogenous nutrient nanoparticles enter the plant through the cracks and crevices between leaf cells called plasmodesmata. The goal is to reach the cell communication pathway, known as the cytoplasm, with the complete mineral requirements plants need to perform essential plant functions such as, cell division (AKA growth), immune defence responses, and reproductive development.

It’s important to note that the deeper into the plasmodesma on the way to the cytoplasm, the stronger the negative ionic charge. Conventional foliar products composed of only one chelated mineral, struggle to achieve efficient nutrient availability, and is why the best foliar products carry a neutral charge. SinC’s homogenous nanoparticles are comparable in size and charge to the best chelated mineral molecules, but it’s homogenous composition of 14 minerals provides unmatched mineral bio-availability for use in plant functions.


Feasibility of Using Mycoherbicides for Controlling Illicit Drug Crops (2011)

Fungal diseases are a normal occurrence where opium poppy (Papaver somniferum) is grown repeatedly. Among the fungal pathogens that can cause serious damage to poppy plants are species of Brachycladium, Crivellia, Fusarium, Peronospora, and Verticillium (Scott 1877 Harrison and Schmitt 1967 Dolgovskaya et al. 1996 Podlipaev et al. 1996 Reznik et al. 1996 Finetto 2008). Crivellia papaveracea and Brachycladium papaveris (formerly known as Pleospora papaveracea and Dendryphion penicillatum, respectively) have received the most attention as parasites that might substantially limit licit poppy cultivation (Milatoviæ 1975a b) and as potential mycoherbicides against illicit opium poppy crop (Del Serrone and Annesi 1990 O&rsquoNeill et al. 2000 Bailey et al. 2000b, 2004a, 2004b UNODC 2002). Both fungi are distributed worldwide wherever poppy is grown (Milatoviæ 1975a Munro 1978 Sivanesan and Holliday 1982).

As noted in Chapter 1, a single fungus may have more than one name, and this can complicate interpretation of the scientific literature. Nowhere is that problem more evident than in the literature on C. papaveracea and B. papaveris. In this report, the publication by Inderbitzin et al. (2006) is used for the taxonomy of those fungi because it used DNA sequence data to identify strains, including many of the ones used in the studies cited in this chapter. Inderbitzin et al. (2006) describe how different authors have used the names in different ways. In the present report, where possible, we have translated the names used by the various authors into the currently accepted names, C. papaveracea and B. papaveris. Readers who consult the original literature may find Table 5-1 useful in attempting to equate the names used in that literature with the names used

here. The attributes of C. papaveracea and B. papaveris are listed in Table 5-2 with the two isolates studied by O&rsquoNeill et al. (2000) as reference strains. When it is unclear which of the two species was studied, we refer to both in this manner: C. papaveracea/B. papaveris.

C. papaveracea/B. papaveris was reported as a pathogen of poppy by Milatoviæ (1975a,b) and Czyzewska and Zarzycka (1960). C. papaveracea and B. papaveris are seedborne pathogens that are widely distributed around the world wherever poppy is grown but are most commonly observed in Europe and Asia (Schmitt and Lipscomb 1975). Both can cause serious damage to poppy when conditions for disease are optimal (Krikorian and Ledbetter 1975 Schmitt and Lipscomb 1975). C. papaveracea/B. papaveris attacks plant roots and parts above the ground disease symptoms include seedling damping-off, girdling of roots, and lesions on leaves, stems, and capsules (Meffert 1950 O&rsquoNeill et al. 2000).

TABLE 5-1 Fungi Used in Various Papaver Mycoherbicide Studies

Fungal Strain (Reference) Identification Given in Reference Identification by Inderbitzin et al. 2006
Strain 7359 (Meffert 1950) Dendryphion penicillatum Crivellia papaveracea
Strain 3 (Meffert 1950) Helminthosporium papaveris Brachycladium papaveris
Isolates used by Del Serrone and Annesi 1990 Dendryphion state of P. papaveracea Strains not studied by Inderbitzin et al. 2006. Inferred to be a heterothallic fungus.
Strain Cf96 (Farr et al. 2000) Dendryphion penicillatum Crivellia papaveracea
Strain Pf96 (Farr et al. 2000) Pleospora papaveracea Brachycladium papaveris
Strain Cf96 (O&rsquoNeill et al. 2000) Dendryphion penicillatum Crivellia papaveracea
Strain B96 (O&rsquoNeill et al. 2000) Pleospora papaveracea Brachycladium papaveris
Strain Pf96 (Bailey et al. 2000a, 2000b) Pleospora papaveracea Brachycladium papaveris
Strain Cf96 (Bailey et al. 2000b) Dendryphion penicillatum Crivellia papaveracea
No strain number inferred to be Pf96 (Bailey et al. 2004a) Pleospora papaveracea Brachycladium papaveris (if the isolate is indeed Pf96)
Strain Pf96 (Bailey et al. 2004b) Pleospora papaveracea Brachycladium papaveris
Strain C-6-3 (UNODC 2002) Pleospora papaveracea (Not studied by Inderbitzin et al. 2006)

TABLE 5-2 Attributes of the Papaver Mycoherbicides Crivellia papaveracea and Brachycladium papaveris

Attribute Crivellia papaveracea Brachycladium papaveris Reference
Reference strain a Cf96 Dendiyphion peniciUatum Pf96 Pleospora papaveracea O&rsquoNeill etal. 2000
Teleomorph (sexual form) CriveUia papaveracea None described b Inderbitzin et al. 2006
Anamorph (asexual form) Brachycladium peniciUatum Brachycladium papaveris Inderbitzin et al. 2006
Sexual reproduction Heterothallic (requires mating partner) Homothallic (self-mating) Inderbitzin et al. 2006
Macroconidiophores c Produced Not produced Inderbitzin et al. 2006
Microsclerotia d Present Absent Inderbitzin et al. 2006: Meffert 1950
Chlamydospores e Not reported Infrequent: intercalary (between apex and base) Fan- et al. 2000: Meffert 1950
Pseudothecia f Present in field material Present in field material: produced by laboratory culnires older than 30 days O&rsquoNeill etal. 2000
Relative virulence Less virulent More virulent Bailey et al. 2000b: O&rsquoNeill etal. 2000

a Strains used by CTNeill et al 2000 and studied by Inderbitzin et al. 2006.

b The situation with B. papareris is unusual because this fungus has a sexual meiotic spore state (Fan et al. 2000). but Inderbitzin et al. (2006) were unable to locate a specimen with the sexual structures to serve as a type for a teleomoiph name.

c Hyphae (vegetative threads) that bear cells that produce macroconidia or large asexual spores.

d Very small rounded mass of hypliae.

e Asexual thick-walled one-cell spores.

f Specialized structures that bear asci (which contain ascospores or sexual spores).

In 1986, C. papaveracea/B. papaveris was evaluated as a potential biological control agent against Papaver rhoeas, a major weed of wheat in Italy (Covarelli 1981 Pignatti 1982). Results of pathogenicity and host-range experiments indicated that C. papaveracea/B. papaveris infected P. rhoeas and reduced its biomass and that it did not infect wheat, maize, barley, sorghum, or oats (Del Serrone and Annesi 1990).

In 1991, researchers at the Institute of Genetics and Plant Experimental Biology in Tashkent, Uzbekistan, recovered a &ldquohighly virulent&rdquo isolate of C. papaveracea/B. papaveris from poppy plants (UNODC 2002). They reported

that the isolate caused 50-75% losses in licit and illicit poppy crops but did not specify under what environmental conditions the losses occurred. Symptoms of disease included damping-off of seedlings and leaf and stem lesions. Poppy capsules and seeds also were affected, and this resulted in smaller capsules and reduced seed production. The discovery of the isolate became the basis of a project on the development of C. papaveracea/B. papaveris as a biological control agent against poppy (discussed in more detail later in this chapter). It is important to note that, particularly for pathogens of the foliage, environmental conditions dramatically influence the amount of damage that a pathogen can cause. Losses can approach 100% under environmental conditions favorable to the fungus, but there might be no infection or damage in conditions unfavorable to the fungus.

In the late 1990s, research on the biological control of poppy (P. somniferum) was carried out at the U.S. Department of Agriculture (USDA) Agricultural Research Service (ARS) laboratory in Beltsville, Maryland, with isolates of C. papaveracea and B. papaveris recovered from poppy plants that were grown in a field in Beltsville. C. papaveracea and B. papaveris were isolated from the same diseased poppy seeds, seedlings, foliage, capsules, and field stubble and from asymptomatic plants (O&rsquoNeill et al. 2000). The two fungi are morphologically distinct, and this makes it possible to distinguish them in culture. Greenhouse and field tests conducted by Bailey et al. (2000b) showed that B. papaveris caused more severe damage on poppy than C. papaveracea. When both fungi were applied in the field, B. papaveris was the more frequently recovered fungus from poppy seed capsules and was the only fungus isolated from the field in the following year (Bailey et al. 2000b). Other studies of B. papaveris focused on whether its efficacy could be enhanced with a phytotoxic protein, Nep1, isolated from Fusarium oxysporum (Bailey et al. 2000a) or the addition of other adjuvants (Bailey et al. 2004b) and on determining the best technique for mass production of its inoculum (Bailey et al. 2004a).

There have been no other publications on the evaluation or development of C. papaveracea or B. papaveris as mycoherbicides against opium poppy since the termination of the project in Uzbekistan in 2001 and the publication of the results of the studies conducted by the USDA ARS in 2000 and 2004.

Three studies can be used as basis for assessing the efficacy of C. papaveracea or B. papaveris as a mycoherbicide agent against opium poppy. A study conducted in Italy was published in the proceedings of a conference (Del Serrone and Annesi 1990), a study conducted in the United States (Beltsville, Maryland) was published in a peer-reviewed journal (O&rsquoNeill et al. 2000), and a study conducted in Uzbekistan (UNODC 2002 report) that was sponsored by the UN International Drug Control Program under the auspices of the UN Office on Drugs and Crime (UNODC) and conducted in 1998&ndash2002 at the Institute of

Genetics and Plant Experimental Biology. The goal of the latter study was to assess the potential of C. papaveracea/B. papaveris &ldquoas an effective, reliable, and environmentally safe biological control agent for opium poppy in realistic field conditions.&rdquo The findings were presented in a report made available to the present committee the report was reviewed by an independent expert and a group of technical experts enlisted by UNODC, but the results have not been published in peer-reviewed journals.

Experiments at the Istituto Sperimentale per la
Patologia Vegetale, Rome, Italy

Del Serrone and Annesi (1990) conducted studies to evaluate the feasibility of controlling a species of poppy, P. rhoeas, with C. papaveracea/B. papaveris isolated from infected P. rhoeas plants in the field. P. rhoeas is an annual weed in cereal crops, especially wheat, in Italy. The committee reviewed this work because of the interest in C. papaveracea/B. papaveris as a mycoherbicide against opium poppy, although Del Serrone and Annesi did not propose to use it to control illicit poppy. Table 5-3 provides a summary of the experimental details.

According to the published report, disease symptoms were observed 3-4 days after inoculation as water-soaking of petiole tissues, followed by drying of the petioles, and finally the wilting and death of leaves. The greatest damage (with over 99% of the leaves infected) was observed in plants that had four to seven true leaves and had been sprayed with a suspension of 1.5 × 106 spores/mL followed by a 24-hour dew period at 25°C. Damage (in percentage of leaves infected) was less severe when inoculated plants were given 12 hours of dew at 23°C (59%) or 24 hours of dew at 15°C (72%). The requirement for 24 hours of dew to cause the most severe damage led the authors to conclude that it is &ldquotoo long&rdquo to consider the use of this isolate as a mycoherbicide. They noted that it would be useful to conduct additional investigations to find &ldquomore adaptable isolates to be used under field conditions&rdquo (Del Serrone and Annesi 1990).

Experiments at the U.S. Department of Agriculture Agricultural
Research Service Laboratory in Beltsville, Maryland

O&rsquoNeill et al. (2000) discovered that P. somniferum seedlings and mature plants produced from USDA plant introduction seed accessions grown in greenhouses and growth chambers in Beltsville, Maryland, were dying of an unknown destructive disease. The pathogen was identified as C. papaveracea/B. papaveris. O&rsquoNeill et al. conducted replicated experiments in growth chambers to determine the pathogenicity and comparative virulence of B. papaveris (isolate B96) and C. papaveracea (isolate Cf96) to poppy plants from three

accessions (White Cloud, Indian Grocery, and Venezuela). The details of the experiments are given in Table 5-4.

The results confirmed that both fungi were pathogenic to the three poppy accessions tested, but B. papaveris B96 was generally more virulent than C. papaveracea Cf96. Symptoms of infection included chlorosis, water-soaking of the stems and leaves, and tissue death. The average disease rating (on a scale of 0-9) on all test plants 7 days after inoculation (DAI) was 8.75-9.00 (93-100% foliage necrosis) with B. papaveris, whereas the same test plants inoculated with C. papaveracea had a rating of 2.75-7.50 (6-12% to 87-93% foliage necrosis) (see Table 5-3). When a similar experiment was conducted with 18-day-old seedlings of White Cloud and Indian Grocery, the seedlings were highly susceptible to both fungi, and necrotic lesions were observed 48 hours after inoculation. Mortality was 100% in seedlings inoculated with P. papaveracea and 97% in seedlings inoculated with D. penicillatum 5 DAI. The seedlings inoculated with B. papaveris were dead, and those inoculated with C. papaveracea exhibited 87-93% necrosis.

O&rsquoNeill et al. (2000) also determined the efficacy of C. papaveracea and B. papaveris in replicated experiments by inoculating poppy plants with different spore concentrations (105, 106, and 107 spores/mL) and then exposing them to different wetness periods by misting them for 0, 6, 12, 24, and 48 hours. The inoculated plants were killed within 9 days when the spore concentration was 106/mL and the wetness period was 24 hours or longer. For B. papaveris B96, at least 6 hours of wetness was required to attain 25-50% foliar necrosis 12 DAI. The efficacy of the spore inoculum increased when the inoculum was formulated with unrefined corn oil White Cloud and Indian Grocery plants inoculated with C. papaveracea or B. papaveris spores with 30% oil and exposed to 6 hours of wetness exhibited 25-50% necrosis 3 DAI (O&rsquoNeill et al. 2000).

TABLE 5-3 Greenhouse Study by Del Serrone and Annesi (1990)

Factors Details
Inoculum C. papaveracea/B. papaveris Spore suspension containing 1.5 × 106 spores (conidia) per milliliter with 0.001% Teepol (detergent) sprayed onto test plants until runoff control plants sprayed with water and Teepol only
Test plants Poppy plants inoculated at four growth stages: cotyledon to three true leaves, four to seven true leaves, eight to 11 true leaves, and 12-16 true leaves
Environmental conditions Inoculated and control plants exposed to different dew periods (6, 12, 18, and 24 h) and temperatures (15, 20, 25, 30°C)
Assessment method Efficacy assessment was based on reduction in plant dry weight and percentage of infected leaves Experiment was performed twice

TABLE 5-4 Growth-Chamber Studies by O&rsquoNeill et al. (2000)

Factors Details
Inoculum Single-conidia isolates of C. papaveracea and B. papaveris obtained from diseased poppy foliage and capsules of plants growing in a field and growth chambers in Beltsville, Maryland
Test plants Poppy plants from Indian Grocery (IG), VEN (from Venezuela, used in opium production), and White Cloud (WC) accessions
Treatments In one experiment (two trials), 8-wk IG, VEN, and WC plants sprayed with B. papaveris isolate B96 and C. papaveracea isolate Cf96 spore suspensions in water (containing 4 × 104 spores/mL) and provided with 100% relative humidity (RH) for 24 h plants then moved to a growth chamber with 40% RH, 28°C/22°C day/night temperature, and 11-h photoperiod
In another experiment (two trials), 18-d-old seedlings of IG and WC sprayed with spore suspensions of isolates B96 and Cf96 in water (containing 3 × 104 spores/mL) and provided 100% RH for 24 h
Different spore concentrations (105, 106, and 107 spores/mL) of isolates and different wetness periods (misting for 0, 6, 12, 24, and 48 h) also tested in growth-chamber experiments
Assessment method Efficacy assessed on disease-severity rating scale (based on visual estimation of percentage of foliage blight) of 0-9 where 0 = 0-3%, 1 = 3-6%, 2 = 6-12%, 3 = 12-25%, 4 = 25-50%, 5 = 50-75%, 6 = 75-87%, 7 = 87-93%, 8 = 93-96%, and 9 = 96-100% necrosis

Experiments in Uzbekistan and Tajikistan

Researchers at the Institute of Genetics and Plant Experimental Biology conducted experiments in 2000-2001 to test the efficacy of fungi that they had isolated from diseased poppy plants in Uzbekistan. Their report refers to the pathogens as P. papaveracea (now referred to as C. papaveracea) and D. penicillatum (now referred to as B. papaveris) (UNODC 2002). Details of the field experiments are given in Table 5-5.

In the trials in Uzbekistan, several formulations were tested, but they were not described in the report. In 2000 and 2001, application of conidia in formulation &ldquo19&rdquo resulted in reductions of 50% and 52% in poppy capsule numbers, respectively, 45% and 63% in capsule weight, and 78% and 81% in seed weight per capsule. Application of formulation &ldquo1&rdquo had similar results in the Uzbekistan trials. In the 2001 trial in Tajikistan, formulations &ldquo24,&rdquo &ldquo25,&rdquo and &ldquo27&rdquo caused the most damage: a 60% reduction in plant height, 60% in capsule numbers, and 90% in plant weight. Formulations &ldquo19&rdquo and &ldquo1,&rdquo which caused the most damage to poppies in Uzbekistan, were not as efficacious in the Tajikistan trial. The researchers thought that the higher ultraviolet-radiation levels at the Tajikistan site, which was 2,500 m above sea level, reduced the efficacy of C. papaveracea/B. papaveris formulations &ldquo19&rdquo and &ldquo1.&rdquo

TABLE 5-5 Field Trials in Uzbekistan and Tajikistan (2000-2001)

Factors Details
Inoculum C. papaveracea/B. papaveris isolated from diseased opium poppy (Papaver somniferum) and wild poppies (P. arrhenium, P. pavonium, P. rhoeas, and P. refracta) in Uzbekistan and Tajikistan
Isolate referred to as C-6-3 used in field experiments because it had &ldquothe greatest virulence and highest growth rate&rdquo
Location and time of inoculation Trials conducted in Uzbekistan and Tajikistan field sites
Inoculations in autumn, at &ldquobudding phase&rdquo of poppy plants
Treatments Inoculum suspension containing 1 × 106 conidia/mL sprayed at 500 L/ha (5 × 1011 conidia/ha) treatments consisted of spores in different formulations identified by numerical designations but of undescribed compositions control treatment consisted only of formulation without spores
Assessment method Efficacy of fungus assessed on basis of reduction in poppy capsule numbers, capsule weight, and weight of seeds per capsule in Uzbekistan trials in Tajikistan trials, efficacy assessed on basis of reduction in plant height, capsule numbers, and plant weight
Final yield data not obtained, because &ldquoit was too dangerous to keep the trial long enough for this&rdquo

According to the Uzbek researchers, inoculation of poppy plants at the rosette stage resulted in leaf infections followed by stem infections and finally plant death within 48 hours of inoculation. Infection at a more mature, capsuleforming stage resulted only in capsule discoloration and stunting. The researchers further reported that under natural field conditions, C. papaveracea or B. papaveris can cause disease in plants at the &ldquobudding phase&rdquo (capsule formation) and that although inoculation at this stage did not cause plant death, the capsule was so affected that it was commercially unusable capsules of infected plants were small, hard, and blackened and contained seeds with lower viability. Laboratory analysis for alkaloids showed reduced concentrations of morphine, codeine, thebaine, narcotine, and papaverine in plants that were inoculated with fungal formulations &ldquo1&rdquo and &ldquo19&rdquo (UNODC 2002). The authors recommended application of the fungus to poppy plants at the rosette stage (before flowering) to destroy the crop (UNODC 2002).

Del Serrone and Annesi (1990) demonstrated that younger plants are more susceptible to damage and that the fungus requires 24 hours of dew to cause severe damage. O&rsquoNeill et al. (2000) also showed that young plants are more susceptible to damage and that C. papaveracea or B. papaveris can cause severe damage or death relatively quickly if the inoculated plants are exposed to a long wetness period, that is, at least 24 hours. However, in a study by Bailey et al. (2000b), field-grown poppy plants from the accession White Cloud inoculated at the rosette stage did not become severely infected until the plants began to flower and form capsules. Bailey et al. postulated that the susceptibility of younger plants in greenhouse experiments was due to the optimal conditions in

the greenhouse, which may not always occur in the field. An important caveat here is that the different research groups worked with different isolates of C. papaveracea or B. papaveris (see Table 5-1).

Results from greenhouse and field studies provide evidence that C. papaveracea and B. papaveris can cause disease on poppy, but the extent of damage or yield loss will depend on several factors, including the virulence of the isolate or strain used, the inoculum formulation, the application rate, the plant growth stage, the poppy cultivar (genotype), and the environmental conditions in the field during and immediately after inoculation.

Mechanisms of Pathogenicity

Pathogenicity is the ability to cause disease disease may result from a pathogen&rsquos infecting, colonizing, and disrupting the normal cellular functions of a plant. C. papaveracea and B. papaveris infect aerial parts of the plant, initially causing chlorotic spots on the leaf, chlorosis on leaf margins, and water-soaking on the leaf and stem, which may be followed by withering and drying of the leaf and the development of stem lesions (Milatoviæ 1975b Del Serrone and Annesi 1990 Bailey et al. 2000b O&rsquoNeill et al. 2000). Infected leaves change from green to gray-green and enter senescence prematurely (Miltoviæ 1975b). Both C. papaveracea and B. papaveris form appressoria, specialized suction-cup-like structures that some fungi require to penetrate plant epidermal cells and cause infection, on poppy leaves, but they can also penetrate the leaf through open stomata (Bailey et al. 2000b). The disease can attack any part of the opium poppy plant&mdashleaf, stem, capsule, root, or seed&mdashat any stage of development (Milatoviæ 1975a). Although diseased seedlings and immature plants might be killed (Del Serrone and Annesi 1990 Bailey et al. 2000b O&rsquoNeill et al. 2000), premature drying of the plant at any of the later stages of development (flowering, capsule formation, or capsule maturity) can greatly reduce the number, size, and weight of the capsules (Bailey et al. 2000b) and the amount of opium that could be harvested from the still-green capsules (UNODC 2010d). C. papaveracea produces sexual and asexual spores on infected poppy tissues (Milatoviæ 1975b O&rsquoNeill et al. 2000) these spores may disperse the pathogen throughout the field and region. Being a seedborne pathogen (Milatoviæ 1975b), C. papaveracea also could be spread through the use of infected seed for planting.

Little is known about the mechanisms underlying the pathogenicity of C. papaveracea or B. papaveris. As noted in Chapter 4, filamentous fungi produce a variety of secondary metabolites (organic compounds produced by an organism that are not required for its immediate survival). Although the function of most secondary metabolites is unknown, many have been shown to have biological activity, including phytotoxicity. The UNODC (2002) report listed several metabolites produced by C. papaveracea that were identified in methanol extracts of fungal cultures and that were found to be phytotoxic in a

detached-leaf assay. Phytotoxicity was measured semiquantitatively (+, ++, +++) over a 48-hour period. Phytotoxins identified included 1, 2-benzene dicarboxylic acid 1, 2-benzene dicarboxylic acid, dipropyl ester 1, 2-benzene dicarboxylic acid, bis (ethylhexyl) ester and two derivatives of tetracosahexaene (squalene). These metabolites have no clear role in disease development (UNODC 2002). Some experiments combining a culture filtrate with the fungus were performed to determine whether fungal efficacy was enhanced by toxic metabolites the results showed little enhancement of the efficacy of C. papaveracea (UNODC 2002). The role of the metabolites in the pathogenicity of C. papaveracea, if any, remains unknown.

Facility, Equipment, and Technology for Large-Scale Manufacture

A general discussion of the fermentation methods used for large-scale production of commercial mycoherbicides is provided in Chapter 4 (see section &ldquoInoculum Production and Delivery&rdquo). C. papaveracea/B. papaveris can be grown to produce infective propagules on a variety of solid substrates and liquid media (Table 5-6). On the basis of studies reviewed in Table 5-6 and the general literature on microbial pesticides, it appears feasible to mass-produce these fungi. The available data on the proposed mycoherbicides provide useful leads but are exploratory any large-scale attempt at production would be more efficient if it begins with basic studies of fungal growth and fermentation, preferably in collaboration with an industrial partner. Such studies are needed to determine the choice of fermentation method (liquid, solid, or biphasic), the type of propagule (mycelium, conidium, chlamydospores, microsclerotia, pseudothecia, or ascospores), the formulation (liquid concentrate, dust, pellet, food-grain-based, or seed-based), the intended delivery method (aerial or groundbased), and the expected efficacy, shelf-life, and handling qualities of the product.

To estimate the amounts of the mycoherbicide product that might be required for a hypothetical program to control illicit opium poppy worldwide, the committee made calculations on the basis of published data on the amounts of inoculum used in field trials (Table 5-7). However, as noted in Chapter 4, the published data are only a guide the actual amounts cannot be determined without testing the finished mycoherbicide product under conditions that simulate operational programs. Typically, this phase of mycoherbicide research and development is done by an industrial producer in collaboration with the mycoherbicide researchers. Therefore, the actual amounts of opium-poppy mycoherbicide required may be higher or lower than the amounts projected in the table. The estimates in Table 5-7 project that large volumes of water would be necessary to apply the opium-poppy inoculum, and this could be an important limiting factor in developing it as a mycoherbicide.

TABLE 5-6 Methods Used for Production of C. papaveracea/B. papaveris Inoculum for Experimental Trials

Reference Method Inoculum Produced
Del Serrone and Anessi 1990 Inoculum produced by culturing C. papaveracea/ B. papaveris on malt extract agar (30% maltose) for 9 days at 25°C with 12-h light and dark periods Spores produced (type not mentioned: presumed to be conidia) used for inoculation
Bailey et al. 2000b: O&rsquoNeill etal. 2000 C. papaveracea/B. papavehs cultured on V8 agar medium for 7-10 days at 23°C with 16-h photoperiod Conidia from agar plate cultures used in laboratory, greenhouse, and field experiments
Bailey et al. 20004b C. papaveracea and B. papaveris cultured on VS agar medium for 6-10 davs at 25°C in the dark Conidia from agar plate cultures used in laboratory, greenhouse, and field experiments
Bailey et al. 2004a C. papaveracea/B. papaveris cultured on agar with molasses, wheat bran, pectin, rice flour, dextrin, cornstarch, soy flour, corncobs, or cottonseed meal, each with brewer&rsquos yeast Greatest amount of radial growth occurred on molasses-brewer&rsquos yeast, soy fiber-brewer&rsquos yeast, and wheat bran-brewer&rsquos yeast agar media: greatest number of conidia produced on soy fiber-brewer&rsquos yeast agar medium: only chlamydospores produced on comstarch-brewer&rsquos veast and dextrin-brewer&rsquos veast aaar media
Bailey et al. 2004a Liquid media (in 100-niL shake flasks) containing dextrin, cornstarch, wheat bran, or soy fiber, each supplemented with brewer&rsquos yeast 1x10 6 colony forming units per milliliter produced on all four substrates after 5 days of growth: chlamydospores (6 x 10 6 mL) produced only in media containing comstarch-brewer&rsquos yeast and dextrin-brewer&rsquos yeast: conidia (10 4 mL) produced only in media containing soy fiber-brewer&rsquos yeast and wheat bran-brewer&rsquos yeast
Bailey et al. 2004a Bench-top fermentation (2.5-L commercial bench-top fermentor) with dextrin (30.0 g) and brewer&rsquos yeast (15.0 g) mixture as medium: medium maintained at 25°C with 200-rpm agitation, fermentation period was 7 days Biomass produced on bench-top fermentor consisted of nonmelanized mycelial fragments and chlamydospores
UNODC 2002 10-mL conidial suspension of C. papaveracea added to 250 niL of liquid medium in flask: inoculated liquid medium (not identified in report) incubated on rotary shaker (70 rpm) at room temperature for 3-4 days, after which mycelia were recovered and spread out on muslin cloth that had been stretched over metal frame: mycelia on muslin incubated at 100% relative humidity for 48 h: spores then collected with vacuum harvester and stored as dry powder Diy spores stored in sealed or nonsealed containers or mineral oil: Tests conducted over a 5-year period indicated minimal reduction in spore viability and virulence: number of propagules produced not discussed in report

TABLE 5-7 Estimated Amounts of the Proposed C. papaveracea/B. papaveris Needed for a Single Application against Illicit Opium-Poppy Crops Worldwide

Reference Amount of Inoculum and Volume of Carrier (Water) Used in Field Trials a Amount of Mycoherbicide (So. Spores) and Volume of Water &rsquoSeeded for Each Application over Worldwide Area b , c , d Amount of Mvcoherbicide (No. Spores) and Volume of Water Needed per Hectare for Each Application
Bailey et al. 2000b 2 x 10 5 conidia niL suspended in Tween 20 or Tween 20 plus com oil. applied at 3 niL Plant or 180-360 L/ha e 6.5 x 10 15 to 13 x 10 15 (6.5-13 quadrillion) spores and 33-66 million liters of water 3.6 x l0 10 to 7.2x 10 10 spores and 182-364 L of water
Bailey et al. 2000a e 5 x 10 5 conidia 111L suspended in water or aqueous adjuvant, applied at 5 x 10 10 conidia.ha in 1.290 L of water (with or without adjuvants) 1.2 x 10 17 (1.2sextillion) spores and 234 million liters of water 6 x 10 11 spores and 1,290 L of water
Bailey et al. 2004b e 1 x 10 6 m/L conidia applied at 8.7 x 10 11 conidia ha in 866 L/ha 1.6 x 10 17 (1.6sextillion) spores and 157 million liters of water 88x10 10 spores and 866 L of water
1 x 10 6 mL cooidia applied at 3.3 x 10 12 conidia ha in 3.300 L ha 6x 10 17 (6 sextillion) spores and 599 million liters of water 331 x 10 10 spores and 3.303 L of water
2 x 10 6 mL conidia applied at 9.9 x 10 12 conidia. ha in 4.950 L ha 1.8 x 10 18 (1.8quintillion) spores and 898 million liters of water 992 x 10 10 spores and 4951 L of water
UNODC 2002 5 g/0.3 m 2 of inoculum composed of fungus-olonized millet seeds: proportion of fungus to millet seed not specified 90 x 10 15 (90 quadrillion) spores and 91 million liters of water 50 x 10 10 spores and 502 L of water

a Amounts of inoculum used in field experiments were not aimed at defining the minimum inoculum quantity needed for effectiveness: this remains to be determined with the actual mycoherbicide product.

b ased on most recent UNODC estimate of total area under opium-poppy cultivation worldwide is 1813 73 ha (UNODC 2010a). These calculations are provided on the basis of a potential worldwide target area to indicate the industrial production capacity that might be needed. The committee regards simultaneous worldwide application or even a worldwide application within a growing season as logistically unrealistic.

c A typical opium-poppy field has 60.000-120.000 plants/ha (see Chapter 3).

d Without knowing the weight of each spore, it is not possible to estimate the required amount in metric tons.

e Descriptions of plot size are incomplete. Estimation is based on the committee&rsquos interpretation of the available details.

Adjuvants and Formulation

For a general discussion of adjuvants and formulations, see the section &ldquoAdjuvants and Formulation in Chapter 4. The use of adjuvants to improve the efficacy of C. papaveracea and B. papaveris against opium poppy was studied by Bailey et al. (2000b, 2004b) and by O&rsquoNeill et al. (2000). Among the adjuvants tested, the most effective were Tween 20 (polyoxyethylene [20] sorbitan monolaurate) and unrefined corn oil. The details and results of the experiments are summarized in Table 5-8.

O&rsquoNeill et al. (2000) showed that the addition of unrefined corn oil to C. papaveracea and B. papaveris inoculum increased the severity of disease caused by these two fungi in White Cloud and Indian Grocery poppy plants. They noted that the formulation of C. papaveracea spores with corn oil and the provision of a 6-hour wetness period rendered C. papaveracea &ldquoalmost as virulent&rdquo as B. papaveris on the tested poppy cultivars.

Researchers in the Institute of Genetics and Plant Experimental Biology also tested various formulations of C. papaveracea/B. papaveris under field conditions, but they identified the formulations only by number and did not provide any information on their composition (UNODC 2002).

The use of a low concentration of Tween 20 as an adjuvant might be costeffective, but the environmental effects of its use might require documentation for registration purposes. The use of unrefined corn oil at 10-30% by volume of the spray mixture is impractical in light of the large spray volumes required (Table 5-6).

TABLE 5-8 Effect of Adjuvants on the Efficacy of C. papaveracea/B. papaveris in Greenhouse and Field Experiments

Reference Adjuvant Tested Efficacy
Bailey et al. 2000b 0.001% Tween 20 in 10% (unrefined) corn oil In greenhouse experiments, application of spores (10 6 /mL) mixed with 0.001% Tween 20 in 10% corn oil resulted in 100% infection by B. papaveris and nearly 100% infection by C. papaveracea B. papaveris with 0.001% Tween 20 in 10% corn oil caused 57.7% mortality in poppy seedlings C. papaveracea with 0.001% Tween 20 in 10% corn oil caused 34.7% mortality in poppy seedlings and 17% of plants sprayed with spores of either pathogen with 0.0001% Tween 20 in water did not exhibit any disease symptoms
O&rsquoNeill et al. 2000 30% (unrefined) corn oil In greenhouse tests, poppy plants inoculated with C. papaveracea and B. papaveris spores mixed with 30% corn oil and provided with a 6-hour wetness period had 25-50% foliar necrosis at 3 DAI
Bailey et al. 2004b 1% Tween 20 In field experiments, B. papaveris spores mixed with 1% Tween 20 caused 68% and 56% necrosis and 22% and 27% reduction in capsule weight per plot within 2 weeks of application in trials 1 and 2, respectively

On-ground application of a mycoherbicide against opium poppy is possible, but from a tactical standpoint, aerial application may be the only feasible delivery method over inaccessible areas. An aerial pathogen such as C. papaveracea/B. papaveris is likely to be most effective when sprayed on the plants (that is, aerial application). However, all the studies that the present committee reviewed have examined only land-based spray application of C. papaveracea/B. papaveris directed at the plants. Of the five papers that the committee reviewed (Bailey et al. 2000a,b, 2004b O&rsquoNeill et al. 2000 UNODC 2002), two identified the tool used to apply the inoculum (Bailey et al. 2000a, 2004b), namely, a Binks spray gun model #15 delivering a spore suspension at 15 lb/in2. Presumably, the other studies used some type of hand-held or backpack sprayer.

Aerial spraying of a liquid formulation of C. papaveracea/B. papaveris is unlikely to be practical, because of the large quantities of water that would be required if the methods used by the researchers were implemented on a large scale (see Table 5-6). Producing the large number of spores required might not constitute a problem, provided that a suitable fermentation method is found, but the availability of water and the ability to transport and apply the required quantities in the field would pose major challenges.

Assessment of Performance

The opium-poppy crop is grown in open fields under full sun, so it should be possible to assess the performance of the C. papaveracea/B. papaveris mycoherbicide with aerial imagery. Although the technology for aerial imagery is available, it has to be adapted and tested for use in measuring mycoherbicide efficacy. As an alternative, on-ground assessment of crop damage combined with interviews with growers could be used however, this approach requires the ability to ensure personnel security and access to targeted regions.

The assessment methods used by the researchers were not developed for use in operational drug-crop control programs. For example, in the field trials conducted in Uzbekistan, the efficacy of C. papaveracea/B. papaveris was assessed on the basis of reductions in poppy capsule numbers, capsule weight, and weight of seeds per capsule. In the Tajikistan trials, efficacy assessment was based on reductions in plant height, capsule numbers, and plant weight. Final yield data were not obtained in these studies, because it was too &ldquodangerous&rdquo to continue the trial (UNODC 2002). O&rsquoNeill et al. (2000) assessed the efficacy of C. papaveracea and B. papaveris in growth-chamber studies with a diseaseseverity rating scale (see Table 5-4). Del Serrone and Annesi (1990) assessed the efficacy of C. papaveracea/B. papaveris on the basis of plant dry-weight reduction and percentage of infected leaves.

The persistence of C. papaveracea/B. papaveris in the environment is an important factor in determining whether the applied fungal strain(s) could potentially affect later plantings of the crop. If the mycoherbicide poses risks to nontarget organisms after release, its prolonged persistence would be a disadvantage rather than an advantage. Thus, as noted in Chapter 4, an understanding of the ability to persist in the environment is an important consideration in proposing the use of a mycoherbicide.

Geographic and Climatic Considerations

Only a few studies provide quantitative information on the survival of C. papaveracea/B. papaveris (Del Serrone and Annesi 1990 Bailey et al. 2000b UNODC 2002). Del Serrone and Annesi (1990) found that moisture on the plant surface for sufficiently long durations (length of time depending on the temperature) at favorable temperatures (10-30°C) are required for spore germination and later penetration of host tissue. All the studies were done at 100% humidity or on wet leaflets.

Bailey et al. (2000b) performed a detached-leaf assay with C. papaveracea and B. papaveris to assess the effect of temperature on their survival and growth on poppy leaf surfaces. Survival and growth were measured in terms of conidial germination, germ-tube growth, and formation of appressoria at various temperatures. Germ-tube growth and appressorium formation were favored at higher temperatures (16-29°C), whereas conidial germination was similar throughout the range of temperatures tested (7-29°C). B. papaveris formed more appressoria than C. papaveracea regardless of temperature and required fewer hours of moisture.

The long-term survival of C. papaveracea and B. papaveris also was assessed as part of field tests of their infectivity (discussed above in the section &ldquoEfficacy and Implementation&rdquo). After tests during the first season were completed, the remaining poppy plant residues in the field were chopped and left on the soil surface over the winter. In the spring, opium poppies were planted in the fields, and plants that had symptoms of disease were tested for the presence of the fungi. Only B. papaveris was isolated from diseased poppy. The investigators suggested that their results could be due to this pathogen&rsquos ability to produce ascospores in the spring (Bailey et al. 2000b).

In the studies in Uzbekistan (UNODC 2002), laboratory and field tests indicated that conidia of C. papaveracea/B. papaveris required the presence of the host plant to survive for longer than 2-3 months. In the presence of host tissue, the pathogen remained viable for up to 15 months when applied to the top 5 cm of soil and for up to 6 months when applied at a depth of 15 cm. In the absence of the host plant, persistence was reduced to 3 months at all depths

during the winter and to 2 months at all depths during the summer. When plant debris was buried in the soil, viable fungi were detected for up to 10 months. However, the UNODC report provided no description of the testing protocols or analytical methods that were used.

As previously stated, such physical factors as unfavorable temperature, moisture, and solar radiation and such biological factors as antagonistic microorganisms in the soil could influence survival of C. papaveracea and B. papaveris, but there are no specific data on the effects of these factors on the two pathogens. Inasmuch as C. papaveracea and B. papaveris already are present in most areas where poppies are grown, the environmental conditions and the life cycle of the plants seem to be such that at least some level of persistence would be achieved.

Transmission and Spread

The potential pathways by which spores and vegetative propagules from a particular application site might move into environmental media are illustrated in Chapter 2 (see Figure 2-1). In general, dispersal of the proposed mycoherbicides after application would depend on the production and natural dispersal of secondary inoculum. Experience with other plant pathogens indicates that short-distance spread of ascospores is facilitated by rain and wind, whereas dispersal of conidia occurs primarily by wind (Li and Kendrick 1995 Hildebrand 2002). Long-distance transport and dispersal by wind and water are possible for infected seeds, infected plant tissues, infested dead plant material, and conidia (Meffert 1950 UNODC 2002). It is also possible for farmers and traders to carry infected or infested materials throughout a region or even into new areas.

The occurrence and susceptibility of the host plant appears to be a major factor in determining the population size of many pathogens, including C. papaveracea and B. papaveris. The pathogens are somewhat host-specific, being capable of infecting P. somniferum and other species of Papaver (Del Serrone and Annesi 1990 UNODC 2002). The pathogens might survive on related hosts or colonize unrelated hosts that are not necessarily susceptible to them, as has been shown with formae speciales of Fusarium oxysporum (see Chapter 4), but there are no data on the presence of C. papaveracea and B. papaveris on or in tissues of nontarget plants. These fungi are widely prevalent across the range where opium poppy is grown (Schmitt and Lipscomb 1975).

No studies are available on the interactions of C. papaveracea or B. papaveris with soil microorganisms or other organisms. In general, as noted in Chapter 4, the presence of competitor or antagonistic microorganisms could reduce the persistence C. papaveracea or B. papaveris. For example, insects and

soil organisms can feed on or suppress plant-pathogenic fungi (Nakamura et al. 1992 Okabe 1993 Suárez-Estrella et al. 2007). Thus, antagonistic microorganisms in the soil could theoretically reduce the likelihood that a C. papaveracea/B. papaveris mycoherbicide would establish populations that are large enough to cause recurrent disease in opium poppy. It is possible that the introduced strains of C. papaveracea or B. papaveris could displace the resident strains but there are no data available to determine the probability of such displacement or its consequences.

Overall, the ecological requirements for the spread and survival of C. papaveracea and B. papaveris cannot be adequately described on the basis of the studies conducted so far. In general, it appears that the pathogens could persist in soil for at least two growing periods in the presence of host tissue. The fact that they are found almost everywhere that opium poppy is grown indicates that the strains distributed are likely to persist at some level once introduced into a site. Although no data are available specifically on the potential dispersal of the fungi from the site of application, it is reasonable to assume that they would spread through infected or infested seeds, plant tissues, or plant debris and by wind and rain.

Microbial pesticides are regulated by the U.S. Environmental Protection Agency (EPA), which requires a variety of tests on the environmental fate and safety of pesticides before they are registered. Chapter 2 and Appendix B describe the types of testing required&mdashincluding product analysis, pesticideresidue analysis, toxicity testing, and toxicity and pathogenicity testing of nontarget organisms&mdashand the assessment of environmental fate. The aforementioned studies have not been systematically performed for the EPA registration of C. papaveracea/B. papaveris. This section reviews the data available on C. papaveracea/B. papaveris in the open literature that are pertinent to an understanding of their potential adverse effects on nontarget plants, animals, and microorganisms.

Effects on Nontarget Plants or Microorganisms

In the UNODC (2002) study, the host specificity of C. papaveracea/B. papaveris was tested against 239 species of plants of economic, medicinal, and ornamental importance, including trees and shrubs, and 52 wild species of plants belonging to 24 families. Some nontarget Papaver species were reported to be susceptible, but none of the other nontarget plants was susceptible. The report provides no names of the plant species and families tested, including the names or numbers of the species in Papaveraceae, and no details of the testing and assessment methods. Hence, the information available cannot be used to assess the risks posed to nontarget relatives of poppy by C. papaveracea/B. papaveris.

Del Serrone and Annesi (1990) tested 14- to 20-day-old plants of &ldquoseveral varieties&rdquo of cereal crops and three Papaver species by spraying them with &ldquothe optimum&rdquo suspension of mitospores (conidia) of C. papaveracea/B. papaveris and growing the plants under &ldquothe best conditions for disease development.&rdquo &ldquoPoppy&rdquo plants were included as a control. None of the cereal-crop varieties tested developed disease by 14 DAI, and the fungus was never reisolated from them. Papaver dubium and P. nudicaule, a wild and a cultivated species, respectively, developed a hypersensitive (resistant) reaction. A few small, black, round spots appeared on the surface of the leaves. Some 30% of the P. somniferum plants had died at 5 DAI. The fungus was not recovered from P. dubium, P. nudicale, and P. somniferum, so there was no evidence that the fungus caused the disease.

Effects on Legal Crop Production

The potential risk to legal production of poppy has not been given any attention in the literature reviewed by the committee. Licit poppy crops may be cultivated for seed, oil, ornamental uses, and pharmaceutical purposes to extract narcotics. It is possible that C. papaveracea/B. papaveris would be present in fields used for legal production. If legal poppy production occurs in or near illicit-opium-producing regions, the inundative release of C. papaveracea/B. papaveris as a mycoherbicide to control the illicit plants could similarly enhance the development of disease in the legal crop owing to drift during application or by secondary inoculum produced on infected plant tissues.

Toxicity to Wildlife, Domestic Animals, and Humans

Although the UNODC (2002) report described secondary metabolites of C. papaveracea/B. papaveris that are phytotoxic, none of the metabolites was tested for mycotoxigenic activity. Two secondary metabolites were identified as derivatives of tetracosahexaene (squalene). Why the authors described the squalene derivatives as &ldquofumonisin-like&rdquo is not clear. Squalene and fumonisins are not derived from the same biosynthetic pathway, so there is no rationale, on the basis of the spectral analyses (mass, infrared, and ultraviolet spectrometry), to conclude that the derivatives are fumonisin-like, and the conclusion that C. papaveracea produces fumonisin mycotoxins is not well supported. The committee found no publications other than the UNODC report on biologically active metabolites, including mycotoxins, produced by C. papaveracea/B. papaveris.

Pathogenicity in Animals and Humans

No reports of human or animal infection with Pleospora, Crivellia, or

Brachycladium were found. A single report on the toxicity of culture extracts of Pleospora for cell lines was reviewed (Ge et al. 2005), but it was not helpful in evaluating the potential risks of infection of humans or animals. If those fungi are shown to be thermotolerant (that is, able to grow efficiently at human body temperature, 37°C), there would be a theoretical risk that increasing their amounts in the environment might lead to infection in immunocompromised humans and animals. However, on the basis of the absence of any case reports, the likelihood appears quite low.

The potential for mycoherbicides to mutate is similar to that of fungi in general, as described in Chapter 4. The diversity of fungal genotypes also is affected by sexual recombination within species. Many fungi that have not been observed to reproduce sexually may do so cryptically, judging from populationgenetics evidence (Taylor et al. 2000). In some cases, population-genetics evidence on sex has led to confirmation by laboratory mating (O&rsquoGorman et al. 2009). New genetic variation can become established in fungal populations by natural selection or by chance. Adaptation to new environments, for example, to new plant hosts or to new cultivars of crop plants can be accelerated by outbreeding and recombination due to sexual reproduction (Goddard et al. 2005 Zhan et al. 2007 Sommerhalder et al. 2010). All those processes could affect Crivellia or Brachycladium species.

However, there is little basic genetic information on C. papaveracea or B. papaveris, so only a generalization about the potential for mutation can be made. There is no reason to expect that the mutation rate of these fungi would be different from that of other filamentous fungi or that they would be more or less susceptible to gene gain, gene duplication, or horizontal gene transfer. C. papaveracea outbreeds by sexual reproduction. B. papaveris reproduces sexually but is homothallic (self-mating) and need not produce recombined progeny (Farr et al. 2000). Thus, adaptation involving virulence or host range could be accelerated by genetic recombination in the case of C. papaveracea but not necessarily in the case of B. papaveris.

Mutation could play a role in determining the toxicity (with respect to secondary metabolites produced) of a mycoherbicide to the extent that mutation results in changes in the amount of toxin produced or the environmental conditions under which the toxins are produced. Concerns about mutationrelated changes in the toxicity of C. papaveracea or B. papaveris are all but impossible to assess because very little research has been performed. As noted earlier, the available data are insufficient to determine what secondary metabolites are produced by C. papaveracea or B. papaveris, let alone in what quantities or how production would be affected by mutations.

According to UNODC, diseases of opium poppy are a normal occurrence in Afghanistan. Farmers report various degrees of damage to their crops in practically all years and regions since UNODC began systematic yield surveys (UNODC 2010d). In the spring of 2010, a fungal disease was speculated to be the possible cause of an opium-poppy blight in Afghanistan. The poppy plants exhibited wilting and other disease symptoms that appeared to be consistent with a fungal infection. Tests of diseased tissues identified two Fusarium species, but they were probably secondary colonizers of the decaying tissue rather than the cause of the disease. C. papaveracea/B. papaveris, which has been linked with past diseases of opium poppy in Afghanistan, was not detected (personal communication, Justice Tetty, UNODC, November 19, 2010), but it was noted that the tissue samples examined were of poor quality (personal communication, Eric Boa, CABI, November 25, 2010).

The UN Afghanistan Opium Survey of 2010 reported that opium production was 48% lower than in 2009 (UNODC 2010d) although the overall area under poppy cultivation remained the same. Disease was considered a major contributor to the reduction in opium yield, but farmers also reported losses due to frost, drought, and pests, such as aphids, other insects, and worms. Poppy capsules were fewer and smaller than in previous years. It is important to note that diseases in major growing areas affected opium poppy plants at the late stage of plant development. The diseased plants were described as exhibiting wilt symptoms with yellowing of leaves, drooping, and finally desiccating completely, all of which are indicative of a collar (stem-root interface) or upper root rot. Those symptoms are consistent with the ones observed previously in the region in connection with fungal infections (UNODC 2010d) but are inconsistent with the typical symptoms of infection by C. papaveracea/B. papaveris. The southern region was the most affected: about 42% of the area under opium cultivation was damaged. The western region was also affected by diseases but to a much smaller degree. In the west, a combination of factors, including frost, played a role, according to farmer reports (UNODC 2010d).

On the basis of the foregoing account, the cause of the reduction in opium production in 2010 in Afghanistan is unknown. Diseases, drought, frost, and pests might have contributed to it. Adverse weather conditions (such as frost and drought) might have predisposed the 2010 crop in different parts of Afghanistan to diseases. Without conclusive evidence based on positive identification of the pathogen, C. papaveracea/B. papaveris could not be implicated in the 2010 Afghan poppy blight epidemic. Therefore, it is not possible to gain any insight from this epidemic to guide the use of C. papaveracea/B. papaveris as a mycoherbicide.


Acknowledgements

The authors wish to thank the family of Abdul Hazdid Harith Tinggal and Ieney Daud for their help and support in all logistical matters during our stay in Brunei. Particular thanks are attributed to Akilah Syafina b. Abd. Hazdid, James M. R. Bullock and Mathias Scharmann, who assisted with much of the fieldwork, and Thomas Endlein for providing Fig. 1. The work was conducted in Brunei Darussalam under the research permit UBD/PSR/5(a), and Nepenthes samples were brought to the UK for SEM under CITES permit BA/MAP/128/0911. The project was funded by a grant from the Leverhulme Trust (F/09 364/G).