New Page 1

Use of Mixtures of Natural Products to Replace Methyl Bromide: Field Studies and Mode of Action as Broad-Activity Pesticides

Oktay Yegen¹ and Sadik Tuzun²

1: Department of Plant Protection, Akdeniz University, Antalya, TURKEY;

2: Department of Entomology and Plant Pathology, Auburn University, Auburn, AL USA

Methyl bromide is extremely important to the Turkish economy- about 43% of the population are agricultural workers, and Turkey uses roughly 1000 tonnes of methyl bromide per year to produce foods it exports to Europe and elsewhere. With the impending phase-out of methyl bromide from use, it is important to Turkey that a viable, preferably non-toxic, alternative be found. We present here data supporting the use of natural plant products as very effective alternatives to methyl bromide or other broad-activity pesticides.

The idea of using natural plant products to combat agricultural pests first occurred to Dr. Yegen in the 1960s, inspired by the use of various wild herbs in traditional folk medicine. Dr. Yegen has followed up on this idea throughout his scientific career, and since 1976 has published articles regarding over 150 plant species which he has surveyed for antifungal and antibacterial activity against plant pathogens. In the 1980s, a serendipitous discovery was made: sealed petri dishes containing plant extracts were found to kill not only the fungi in the dishes, but all the other fungi in the incubator as well. Clearly, the antimicrobial agent was very potent, and active in vapor form. From this point onward, Dr. Yegen and his collaborators have been investigating the properties of mixtures of natural plant products and their uses as alternatives to pesticides and fumigants such as methyl bromide. “Natural plant product” is used to mean either extracts taken from one plant species, or a mixture of extracts from a variety of species. In the authors’ experience, mixtures of extracts are generally more effective.

Extensive laboratory tests have shown that even as little as 25 ppm of plant product per volume air, when placed as a droplet on the lid of an inverted petri dish inoculated with either a phytopathogenic fungus (including Phythophthora capsici and Rhizoctonia solani) or bacteria (including Erwinia amylovora), is adequate to retard or even entirely suppress the growth of these microorganisms. Microscopy experiments have demonstrated that fungal hyphae exposed to inhibitory concentrations of these vapors thicken, and cease to grow. If the plant compounds are removed, the fungi are still unable to grow normally, indicating that the damage done by exposure to the vapor is not reversible. Sporulation of some fungi, notably Penicillium species, was also affected by exposure to the vapor.

During the conference presentations, the lysis of P. capsici zoospores and the death of Meliodogyne incognita individuals upon exposure to the vapors of a plant extract solution were demonstrated via a real-time video of a microscopy experiment. A sealed glass chamber was constructed on a microscope slide, connected at each end via tubing to a stoppered flask, in which a 100 ppm aqueous plant extract solution was stirred. The microscope slide came into contact with only the vapors from this solution. The vapors were sufficient to lyse zoospores within one minute, or to kill M. incognita within 9-10 minutes of continuous exposure. The nematicidal effects of the natural plant compounds within a natural soil environment have also recently been verified.

The effect of these natural compounds against P. capsici under greenhouse conditions is summarized in Figure 1. Even as little as 100 ppm of the natural plant products added to the soil is sufficient to provide protection superior to that of Dazomet, the currently recommended alternative to methyl bromide. These experiments have been replicated extensively in multiple laboratories, with consistent results. Natural plant products also outperform Dazomet in field trials. Figure 2 summarizes data from two separate field studies, conducted in soils naturally infested with a variety of pathogens and pests, including phytopathogenic fungi and nematodes. The results clearly indicate that not only do natural plant products suppress disease and promote healthy plant growth, they also are able to perform consistently under field conditions. It should be noted that, for the field trials, the natural plant products were added to the soils via an aqueous spray- irrigation equipment is sufficient to distribute them. It should also be noted, that, in field study II depicted in Figure 2, the natural products, applied with one week of solarization, produced results similar to a commercial application of methyl bromide combined with the prescribed solarization (data not shown).

Based on these and other findings, four different products, based on different mixtures of plant extracts, have been developed and are currently in the process of being patented. BIOFUME and BIOFOL are products with slightly different spectra of activities, formulated for application to soils. BIOBUG is formulated for foliar applications, and BIOSTORE is formulated for storage applications. These active ingredients of these products are derived from plants used traditionally used in Turkey for medicinal purposes, and when appropriately diluted, have no known toxicities against human beings.

The effects of these products on the natural soil microflora is a matter of concern- do these products interfere with the growth of plant-beneficial microorganisms in soils, for example, and do these products persist and accumulate in soils and in food chains? BIOFUME has no obvious net negative effect on the soil community. An initial decrease in the quantities of various soil microorganisms (including bacteria, actinomycetes and fungi) is quickly reversed and the soil returns to a condition similar to that of untreated soil (Figure 3). Figure 4 illustrates the effects of BIOFUME and Dazomet applications on the soil microfloral respiration, as measured via soil dehydrogenase activities. Respiration is clearly hindered by Dazomet, but not by either application of the BIOFUME product.

It is very clear that exposure of pathogens and pests to the volatile ingredients of these mixtures has a direct pesticidal effect, but what happens when the vapor disperses? As Figure 2 demonstrates, natural plant products (the BIOSOL formulation, in this case) are able to effectively control diseases in natural soils that contain a wide array of indigenous microflora. The authors were able to isolate a Pseudomonas fluorescens strain (TR97) from natural soil to which natural plant products had been added. Interestingly, this isolate was able to metabolize the natural plant products. P. fluorescens TR97 also demonstrated substantial activity against phytopathogens: when the bacterium was inoculated into soils alone, or in combination with natural plant products, the result was a uniformly high (over 90%) control of disease. Natural plant extracts are therefore able to modify the composition of the soil microflora in a manner that appears to benefit the plant. If microorganisms are present in the soil which can degrade the plant extracts, the extracts themselves won’t necessarily last very long in soil- in fact, one week after the addition of BIOFUME to a soil, the authors were unable to recover any trace of it. It is possible that these natural plant products protect plants both directly, by killing pathogens and pests upon immediate application, and indirectly, by enhancing the number of plant protective microorganisms in soil so that pathogens cannot re-establish themselves later on.

Recently, 100 ppm per volume applications of BIOFUME, directly added to soil (a commercial potting mix), were found to protect cabbage plants against the leaf pathogen Xanthomonas campestris more effectively than 100 ppm per volume spray applications made directly to the leaves. It is speculated that either a component of the BIOFUME product enters the plant via the roots, is transported systemically and is able to inhibit pathogen growth in the leaves, or else BIOFUME may somehow be able to induce systemic plant defense responses that would limit pathogen infections. Further work is required before any conclusions can be drawn.

All the Naturale-Agro natural, non-toxic pesticide products are based on extracted materials from at least 8 different plant species, which when analyzed yielded at least 60 different chemical compounds. Of these, at least 14 have pesticidal activities. These 14 different compounds may be active by themselves, or their activity may synergistically increase when combined, and these compounds do not all necessarily act on the same site on a given pathogen or pest. These natural plant product mixtures, therefore, act like multi-site pesticides- they have a broad spectrum of activity and it is difficult for pathogens or pests to develop resistance to them.

The results of the authors’ many laboratory, greenhouse and field studies clearly demonstrate that the natural extracts obtained from particular plants, their volatile phases in particular, can effectively kill soil pathogens without causing obvious long-term harm to populations of beneficial microflora. In fact, populations of plant-beneficial organisms, which may suppress pathogen proliferation in soils, may be increased by the application of natural plant products. These natural extracts can and should be used together with solarization: since the active compounds are thermostable, increased temperatures will increase the volatility and distribution, and therefore the activity, of these compounds within the soil. The authors have also demonstrated that these compounds do not persist in soil, and are subject to biodegradation. The active ingredients have no known toxicities to humans when used in diluted form, and the plants from which the extracts are obtained have been used in traditional forms of medicine for centuries, without any reported illnesses or side effects resulting from their use. The Naturale-Agro natural, non-toxic pesticide products are economical, with production costs similar to those of methyl bromide. Being both effective and economical, these natural plant products are a realistic alternative to methyl bromide that can be embraced even by developing countries that cannot afford more costly pesticides. Moreover, since the plants used in the preparation of these extracts will be farmed (not wildcrafted) on erosion-prone soils not suitable for agriculture, the production of these natural pesticides will help reduce erosion in Turkey and provide new sources of revenue for local economies.