การผลิตเชื้อรา Biocontrol Trichoderma และ Beauveria

This article is from ECHO Asia Note #20

Introduction

In nature, dozens of species of harmful fungi can quickly kill a plant, including Fusarium spp., the causal agents of Fusarium wilt, and Phakospora pachyrhizi, the causal agent of soybean rust (Figure 1). Fungi

are unable to produce nutrients on their own, so they must find another source; sometimes that source is old bread, orange peels, a rotting tree trunk, or a plant’s translocation tissues. These pathogenic fungi thrive in conditions of poor air circulation, slow water drainage, over-irrigation or too much rainfall. Such poor conditions can often be prevented by spacing plants properly, following an irrigation schedule, and removing fungus-prone debris, such as old plant material and weeds. No matter what we do, though, there is a good chance that pathogenic fungi will infect our plants at one time or another.

Unfortunately, in our modern world, chemical fertilizers and pesticides have become the norm in agricultural production, causing severe and serious environmental pollution. Use of these agricultural methods can lead farmers to become dependent on more and more inputs, as environmental imbalance ensues.

Fortunately, Fungi Kingdom is not exclusively populated by pathogenic intruders, dwelling unpoliced in the murky corners of the invisible world. Two particular beneficials of the fungal world, Trichoderma spp. and Beauveria bassiana, have been widely studied for their beneficial properties in agricultural production. The potential of these fungi species is especially exciting because of their ability to improve agricultural productivity while decreasing the development of fungicide-resistant pathogens (Studholme, 2012).

Beauveria bassiana

Beauveria bassiana is a common soilborne fungus occurring worldwide. It is an insect-pathogenic fungus. When spores of B. bassiana contact the shell of an insect, they penetrate the exoskeleton and begin

producing a toxin called Beauvericin, which weakens the host’s immune system until the insect dies (Caldwell, 2013). Eventually, the fungus will fill the entire body cavity. Under high humidity conditions, the fungus will grow through softer body parts, creating a characteristic “white bloom” appearance as the fungus covers the body (Figure 2). The whole process is slow, happening over three to seven days, so it takes time to suppress the insect population and a single application would not be sufficient. Also, note that Beauveria will only suppress and not eradicate an insect population, killing between 50 and 75 percent of the population on average. Spraying during times of higher humidity and at earlier insect life stages will increase effectiveness (Caldwell, 2013).

Beauveria is recommended mainly for the control of chewing pests, white fly and beetles. Since contact with the pest is required, it can be applied at any time without great risk to bee populations, as the hive is elsewhere. Even so, avoid spraying at times when bees are most active. In addition to controlling agricultural pests, certain strains of Beauveria are effective against bed bugs and termites. Before you purchase a commercial strain, do some research or read the label to find out what that strain is effective against. While most research has been done on its insecticidal activity, some studies have shown Beauveria to act against soil fungi such as Rhizoctonia, a pathogenic fungi that causes damping off, root rot and many other pathogenic conditions in a wide variety of plants (Ownley, 2004).

Trichoderma sp.

Trichoderma sp. is a genus of fungi also commonly occurring worldwide, with different species native to different areas; hence the ‘sp.’ designation above. The species are frequently found isolated from

agricultural soils or are seen as green spots on tree bark and deteriorating wood (Figure 3). Several strains of Trichoderma have been developed as biocontrol agents. As a decomposer, Trichoderma fungi can be used to help speed up compost piles. Trichoderma sp. are most effective as a seed treatment, to prevent death from other fungi in emerging and young plants; in this role, Trichoderma can offer improvement over chemical seed treatments. Both encourage germination, yet Trichoderma subsists in the soil and the plant, promoting long-term benefits that a chemical treatment cannot (Harman, 1997). As a soluble spray, Trichoderma is also beneficial when applied to flowers, to prevent fungal growth that decreases fruit set.

Until recently, the benefits of the Trichoderma fungus was thought to be from its ability to produce enzymes that target and degrade chitin, the structural component in the cell walls of fungi and insects. However, recent research has shown that the diverse benefits of Trichoderma are caused by different mechanisms (Contreras-Cornejo, 2009). It has been shown to enhance general plant biomass production and lateral root growth, through interactions that promote auxin production in the host plant. Auxins are a class of plant hormones integral in plant growth and development. Trichoderma is able to form a symbiotic relationship with some plants, even living inside the plant’s vascular system and between cells (Hermosa, 2011), helping the plant resist foliar fungal pathogens (such as rice blast on rice plants) (Studholme, 2012). The fungus can also help plants better tolerate abiotic stress compared to non-inoculated plants (Hermosa, 2011). However, its anti-fungal ability can make Trichoderma harmful in the vicinity of mushroom production.

Producing Beauveria and Trichoderma

Strands of both Beauveria and Trichoderma fungi have been studied in lab conditions. Strains with the most effective beneficial properties have been isolated and reproduced, and are commercially available in Southeast Asia. Enzymes produced by these fungi differ depending on the strain, making their impact potentially unpredictable (Contreras-Cornejo, 2009). For this reason, we do not recommend that you attempt to find and reproduce your own ‘local’ variety. We do recommend, however, that you work to create soil conditions that will promote the growth of these beneficial fungi in your soil. You can do this by using minimal tillage or no-till techniques, by mulching, and by not spraying chemical fungicides.

Fortunately, producing your own Beauveria or Trichoderma spray at home is possible and quite easy. First, purchase a commercial strain, usually available as a dry powder. Be sure to keep this as your starter source for growing more batches of the fungus. Fungi can evolve easily over many generations; if you continually inoculate your substrate with spores from your previous batch, after only a few generations you may have something quite different (and much less effective) than the original. It is worth the cost to use the commercial strain each time you create a new batch.

Supplies

Be careful to avoid getting any fungus, no matter how beneficial, into

your lungs or eyes. We recommend wearing goggles, a dust mask and gloves when inoculating the growing media. Beauveria, in particular, can affect humans, but only in extraordinary circumstances. All four reported cases have occurred when the host’s immune system is already severely compromised.

Other supplies that you will need include: clean rice or sorghum, water, a rice cooker and spoon, large clear plastic bags, rubber bands, a needle, and your Trichoderma or Beauveria powder (Figure 4).

Procedure

1. Mix three parts rice to two parts water (3:2) in the rice cooker. Sorghum can be used in place of the rice, if it is cheaper or more readily available. Make enough to fill your rice cooker, and turn the rice cooker on.
2. When the rice has finished cooking, place two to three large spoonfuls (serving spoons) of cooked rice into a new plastic bag. Pack rice down, then flatten the bag and fold the bag over itself to prevent air (containing foreign spores) from getting in while the rice cools. Let the rice cool until it is comfortable to hold against skin.
3. Open the bag and sprinkle ½ teaspoon of Trichoderma or Beauveria powder on the rice (Figure 5). Close the bag and seal tightly with a rubber band at the top of the bag (Figure 6). Don’t try to force all the air out of the bag. Mix the rice around to spread the spores among the rice, then pack the rice down again.
4. With a needle, poke 10-15 holes in the upper part of the bag where there is no rice. The holes will allow for air exchange. Alternatively, a few changes can be made to the procedure to reduce the likelihood of contamination. Instead of sealing the bag with a rubber band and poking holes in the bag with a needle, you can thread the bag through a 3 cm section of thin PVC pipe and use cotton balls or shreds of cloth to fill the hole. The cotton balls or shreds of cloth will allow for air exchange while preventing contamination, which is more likely with the holes in the rubber-banded bags (Figure 6).
5. Store the bags in a clean (preferably disinfected) indoor location at room temperature. Store in a an area with natural or artificial lighting.
6. After two days, mix the rice again inside the bag and pack it down.

   

    

7. After seven days, the fungi should have taken over the whole bag. It is now usable, but can live in the bag for three to four weeks longer. Healthy Trichoderma should have a sweet coconut odor and is most often dark green (Figure 8) but can be white or light yellow. Beauveria is scentless and should be white (Figure 6).

Your bags are contaminated by other fungi if you see different colors, especially black, and if the smell is putrid. Don’t use any of the contaminated mixture, even if part of it looks pure. Discard the whole bag.

If problems persist, try completing the entire process under a biohood or a similar biosanitary machine. Directions for building a homemade biohood are available from ECHO on ECHOcommunity.org (Figure 7).

For a different method of inoculating corn with Trichoderma, see “PhilRice,” under “Other Helpful Sources” below.

Using Beauveria and Trichoderma

When your Trichoderma or Beauveria bags are finished, dilute 1 kg of inoculated rice into 200 liters of water* to make a sprayable solution. Make sure to rinse the bag and rice clean to get all the possible spores. Separate the rice from the liquid. Once the Trichoderma or Beauveria is mixed with water it must be used or discarded. Mixed solution will not remain effective long term.

Spray every three or four days while pests persist, especially on the underside of leaves. Sprays are especially effective when insects are young, during high humidity and when the spray is highly concentrated with spores. You can continue to spray once per week when the fungus or insect pest is under control. Do not mix with other sprays and avoid spraying other products for four days before or after spraying Trichoderma or Beauveria (Caldwell, 2013).

The Trichoderma-inoculated rice can also be added straight to compost piles, potting soils or planting stations (three days before planting).

Conclusion

“Natural Farming” methods encourage the use of nature’s processes to replace potentially harmful chemical or inorganic pesticides, fungicides and fertilizer. Many of these methods are not yet widely recognized in Western agriculture or in the academic realm. However, the effects of the fungi Trichoderma and Beauveria as beneficial biocontrol agents are widely researched and are commercially available worldwide. Both fungi are easy to reproduce at a low cost, and their potential makes them deserving of the attention they are receiving in the academic world and among farmers in Asia.

*This is the rate used by Maejo University, Chiang Mai, TH: Boonsong Thansrithong, Agriculture Program Manager, ECHO Asia

Works Cited

Caldwell, B., Sideman, E., Seaman, A., Shelton, A., Smart, C. 2013. Resource guide for organic insect and disease management, 2nd edition. Cornell University, Ithaca, NY: Arnold Printing Corp.

Contreras-Cornejo, H., Macias-Rodriguez, L., Cortes-Penagos, C., Lopez-Bucio, J. 2009. Trichoderma virens, a plant beneficial fungus, enhances biomass production and promotes lateral root growth through an auxin-dependent mechanism in arabidopsis. Plant Physiology, 3(149), 1579-1592. doi: PMC2649400

Harman, G. 1997. Trichoderma for biocontrol of plant pathogens: from basic research to commercialized products. Informally published manuscript, Departments of Horticultural Science and of Plant Pathology, Cornell University, Geneva, NY.

Hermosa, R. 2011. Plant-beneficial effects of Trichoderma and of its genes. Microbiology,158(1), 17-25.

IPM Thailand. 2013. Trichoderma. 
Langle, T. Beauveria bassiana (bals.-criv.) vuill. – a biocontrol agent with more than 100 years of history of safe use. Agriculture and Agrifood Canada, Pest Management Center. 

Mercure, P. 1998. Vascular wilts of tomato. Department of Extension, University of Connecticut, Storrs, CT.

Ownley, B., Pereira, R., Klingeman, W., Quigley, N., Leckie, B. 2004. Beauveria bassiana, a dual purpose biocontrol organism, with activity against insect pests and plant pathogens. Emerging concepts in plant health management 2004 (pp. 255-269).

Studholme, D., Harris, B., Le Cocq, K., Winsbury, R., Perera, V., Ryder, L., Ward, J., Beale, M., Thornton, C., Grant, M. 2012. Investigating the beneficial traits of Trichoderma hamatum gd12 for sustainable agriculture—insights from genomics. Frontiers of Plant Science, 4(258), doi: PMC3726867

Surendra, D. 2013. An update on the bagrada bug. Unpublished raw data, Department of Agriculture and Natural Resources, University of California.

Other Helpful Sources

Hayes, Dr. C. 1998. Trichoderma harzianum, strain T-22. Department of Entomology, University of Wisconsin, Madison, WI. Biological Control News.

Kasetsart University Research and Development Institute. 2012. Useful of Trichoderma.

Mahr, S. 1997. The entomopathogen Beauveria bassiana. Department of Entomology, University of Wisconsin, Madison, WI, Biological Control News.

PhilRice. “Trichoderma: Biofungicide for vegetables.” Rice Technology Bulletin No. 62: 18p., October 2009.

Samuels, G.J., Chaverri, P., Farr, D.F., & McCray, E.B. 2014. Trichoderma. Online, Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved January 7, 2014. 

Samuels, G.J. United States Department of Agriculture, Agriculture Research Service. 2000.Trichoderma stromaticum sp. nov., a parasite of the cacao witches broom pathogen.