Final Report for GNE14-084
Present research investigated an alternative method of crop improvement through the usage of biological control agent, Trichoderma. Filamentous fungi in the genus Trichoderma are robust biological control agents as they utilize several modes of action including resistance, antibiosis, competition and myco-parasitism. We demonstrated the ability of Trichoderma-derived biostimulatory gases to stimulate plant growth. Plants, tomatoes and Arabidopsis, exposed to Trichoderma volatile organic compounds (VOCs) exhibited growth promoting effects including increased plant biomass and robust root growth. We evaluated several nutrient conditions to optimize Trichoderma VOC production and measured plant responses to changes in the volatile production. Individual compounds were screened using Arabidopsis and further developed to be used in foliar application in tomatoes. Of the compounds tested, we identified several that showed significant increase in plant fresh weight and total chlorophyll concentration. We also identified solvents that can be safely used on tomatoes for greenhouse application.
The genus Trichoderma is one of the most widely researched genera of filamentous fungi with numerous applications in agriculture, industry and the environment. Trichoderma species are known producers of secondary metabolites with agricultural significance as they often exhibit antifungal and antibacterial properties. Trichoderma species are robust biological control agents because they utilize several modes of action including resistance, antibiosis, competition and myco-parasitism. Since Trichoderma possess innate resistance to many chemicals used in agriculture such as fungicides, they are readily used as part of the integrated pest management practices. While the association between plants and Trichoderma is classified as symbiotic they have the ability to reduce plant diseases and promote plant growth and productivity.
Trichoderma species are prolific producers of volatile organic compounds (VOCs). There is increasing evidence that some Trichoderma volatiles are bio-stimulatory and have the potential to enhance plant growth and development, including tolerance to biotic and abiotic stresses and induction of plant resistance to pathogens. We have previously demonstrated that Trichoderma VOCs induced growth promoting effects in plants. Exposing Arabidopsis to Trichoderma VOCs caused increased plant biomass, robust root growth and lessened the effect of abiotic stressors like low light and extreme temperatures. The exploitation of Trichoderma volatiles as plant growth promoters has the potential to become a powerful tool in agriculture. The overarching goal of this research was to develop ways to improve the sustainability of greenhouse vegetable production by developing methods that allow growers to use the inexpensive biological control agent Trichoderma and its biostimulatory gases in vegetable production thereby reducing fertilizer needs, and using water and energy resources more efficiently. The research focused on evaluating nutrient conditions to optimize biostimulatory gas production by Trichoderma and identify specific compounds that can be directly applied to plants as a foliar application. By increasing the production of biostimulatory compounds by Trichoderma, we can accelerate the growth of plants, improve yield and increase overall production of vegetables in the greenhouse.
1. Evaluate nutrients to optimize biostimulatory gas production by Trichoderma.
a) Assess the effectiveness of nutrients for optimal fungal volatile emissions using split plate bioassay, b) Evaluate optimized nutrient condition for best stimulation of tomato seedlings.
2. Identify specific fungal volatile organic compounds that are responsible for plant growth promotion. a) Expose chemical standard compounds in low concentrations and evaluate biostimulation and/or phytotoxicity on plants, b) Expose tomatoes to growth promoting compound(s)
3. Develop a method of application of biostimulatory volatile compound(s). Evaluate solutions for tomato seedling application.
4. Provide outreach through professional meetings, publications, undergraduate education, and training in agricultural extension.
Evaluate nutrients to optimize biostimulatory gas production by Trichoderma.
Trichoderma viride was grown in 35x10 mm Petri dish on 4 ml of fungal media and incubated for five days at 27 +1 ?C in high humidity prior to the start of the volatile exposure experiments. Several fungal nutrient conditions were screened: malt extract agar (MEA), potato dextrose agar (PDA), yeast extract agar with sugar (YESA), Czapek’s Dox agar (CZA), and oatmeal agar (OAT). All media were purchased from Difco (Detroit, MI, USA).
Arabidopsis thaliana seeds (ecotype Columbia-7) were surface-sterilized using ethanol and bleach solution. Five surface sterilized seeds were sown onto a 100 x 15 mm partitioned petri dish (split- or I-plate) containing Murashige and Skoog medium with vitamins, 3% sucrose, 0.03% phytagel (pH 5.7). Seeds were stratified at 4?C for three days prior to volatile exposure.
For the double plate-within-a-plate system, plate containing Trichoderma was placed into the partitioned Petri dish containing the stratified Arabidopsis seeds, or one week old plant seedlings. Plant and fungi were grown together in a growth chamber at 23 +1 ?C with a 16-hour photoperiod for 14 days. For controls, the same plate-within-a-plate system was used with sterile media. At the end of the exposure period, the plants were removed from the exposure conditions; the shoots were separated from the roots, and weighed before the total chlorophyll concentration was determined.
Seeds of tomato (Solanum lycopersicum L. cv. Ponderosa) were purchased commercially. Seeds were surface sterilized sown onto a 473 ml volume sterile culture vessel containing 100 ml of MS media. To expose tomato seeds, a plate (35 × 10 mm) containing the Trichoderma culture was placed inside a sterile foil container (50 ml volume). The foil containing the fungal plate was then placed inside the culture vessel containing sterilized seeds. Tomato seeds were germinated in the presence of Trichoderma VOCs and grown together in a growth chamber with 16-hour photoperiod at 25±1°C for 21 days. At the end of the exposure period, tomato seedlings were removed from the exposure conditions, photographed, and the fresh weight of plant shoots and total chlorophyll content were measured.
Total chlorophyll concentration of plants exposed to Trichoderma VOCs was determined by submerging fresh shoot tissue overnight in 1 ml of 80% acetone in the dark at 4?C. The total chlorophyll concentration (chlorophyll a and b) was calculated from the equation, ((8.02)(A663) + (20.2)(A645))V/1000W, where V is volume and W is plant fresh weight. The chlorophyll data were expressed in relation to the fresh weight of the plant shoot. Root hairs and leaf tissues were examined using phase contrast microscopy to detect general cell size, stomata numbers and root hair nucleus placement.
Three replicates were used per treatment conditions and the experiments were repeated three times. Quantitative results were expressed as standard error of the mean and analyzed using Excel software, SigmaPlot (SPSS Science Inc., IL), and/or R. Student’s t-test and one-way analysis of variance (ANOVA) between groups were performed for all quantitative data.
Identify specific fungal VOCs that are responsible for plant growth promotion.
Chemical standards were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) to allow for the introduction of defined concentrations. Following compounds were evaluated: 6-amyl-alpha-pyrone, 2-methyl butyraldehyde, 2-ethylhexanal, octanoic acid, 2-methyl-1-butanol, 2-pentanone, isobutyl alcohol, 3-methyl-1-butanol, 1-decene, (-) Limonene, (R)-(+)-Limonene, octanol, 2-n-heptyfuran, trans-2-octenal, 1-octene, (S)-(-)-2-methylbutanol, 1-octen-3-one, nonanal, octanol, 3-octanone, 2-heptanone, butyraldehyde, and 1-butanol. We also used 1-octen-3-ol, a compound previously identified to induce phytotoxicity, as another control. We exposed 1 parts per million (ppm) and 0.5 ppm concentration to seeds and 14-days-old plants.
For the germination assay, a Petri dish containing 50 surface sterilized seeds was placed into a one liter glass tissue culture jar. An appropriate aliquot of each compound was added, volatilized, and the jar was sealed with a translucent polypropylene screw cap. The seeds were exposed to the compound for 72 hours in a growth chamber at 23 + 1 ?C with a 16-hour photoperiod. The control seeds were exposed to the same conditions without the addition of VOCs. At the end of the exposure, the seeds were removed and examined visually using light microscopy. The seeds were scored into three categories, no germination, visual germination (presence of radical), and seedling formation (presence of radical, hypocotyls, and cotyledons).
For vegetative plants, five plants were grown in a growth chamber at 23 + 1 ?C with a 16-hour photoperiod for 14 days following stratification. A Petri plate containing 14-day-old plants was placed into a glass tissue culture jar and an aliquot of the compound of interest was added, volatilized, and the jar sealed and placed in the growth chamber for 72 hr. The plants were removed from experimental conditions, observed for morphological features, individual plants weighed, and total chlorophyll content obtained.
Several compounds that increased Arabidopsis biomass were tested on tomatoes at 10 ng. Low concentration of individual compound was added into the phytotray containing 14 day old seedlings and allowed to grow for several days. At the end of the exposure period, individual plants were assayed for fresh weight (FW), dry weight (DW), water content (WC=(FW-DW)/FW x 100), total chlorophyll concentration (stated in 1a), and shoot and root tissue were observed using microscopy.
Develop a method of application of biostimulatory volatile compound(s).
Evaluated solutions for tomato seedling application in greenhouse. Several solvents were tested on tomato seedlings to evaluate phytotoxicity. Compounds isopropyl myristate, (-)-trans-caryophyllen, ocimene, methanol, and ethanol were tested for phytotoxicity. Plant growth promoting compounds identified previously were dissolved into ethanol and methanol (10 ng solution). Tomato seeds were sown onto sterile soil and allowed to germinate under normal conditions. Following germination, seedlings were sprayed every 72 hours for 14 days. Following exposure, seedlings were measured for fresh weight and total chlorophyll concentration.
The results demonstrate that Trichoderma volatile-induced plant growth promotion differs pending on several parameters. The degree of plant growth promotion or inhibition in Arabidopsis was dependent on the environmental and physiological conditions of the fungi and plants (Figure 1). In addition, we discovered that Trichoderma-derived VOCs induced similar plant growth promotion in tomato, with a significant increase in plant biomass, larger plant size, and significant development of lateral roots (Figure 2 and 3). This suggests that other economically important crop plants will most likely respond similarly to the presence of biostimulatory gases.
Trichoderma viride VOC-induced growth of Arabidopsis led to an increase in plant biomass (41%) and chlorophyll content (89%). In addition, similar responses to VOC mixtures were obtained in tomatoes, i.e. a significant increase in plant biomass (>99%), larger plant size, and significant development of lateral roots, suggesting that plant growth promotion may occur through a similar mode of action in different types of plants. The volatile exposure induced increased lateral root development and very active root hair growth as indicated by increased cell size and nucleus location. The effects were similar to those found in direct interaction with the fungus.
Several fungal media were screened using Arabidopsis for optimal plant growth promoting volatiles. Arabidopsis and tomato plants responded well to fungi grown on MEA, OAT, and CZA (Figures 4 and 5). However, fungi grown on OAT media grew aggressively due to nutrient rich condition leading to an increase in contamination and reproducibility issues. Tomato seedlings exposed to MEA grown Trichoderma had a 99% increase in fresh weight and chlorophyll while those exposed to OAT grown fungi had a 160% increase in fresh weight and 140% in chlorophyll. However, the increase of fresh weight and chlorophyll was not significant between the two nutrient conditions.
Twenty four chemical standard compounds produced by Trichoderma were evaluated for their effects on A. thaliana seeds and vegetative plants in order to determine if individual compounds could mimic the effects of Trichoderma volatile mixtures on plant growth. At the end of 72 hour exposure period, seeds grown in the absence of compound germinated fully and formed seedlings at 82%. A seedling has a root, hypocotyl, and fully green expanded cotyledons. Seeds exposed to 1-octen-3-ol exhibited severe phytotoxicity, where 50% of the seeds did not germinate and 50% arrested at germination (root protrusion only) and did not progress to seedling development. Other less inhibitory compounds induced delayed seedling formation where the cotyledons emerged but did not exhibit full cotyledon expansion and the cotyledons were white or pale green. Compounds 6-amyl-alpha-pyrone, octanoic acid, isobutyl alcohol, 1-decene, limonene, and 2-n-heptyfuran had comparable germination and seedling formation rate. Of these compounds, seeds exposed to 1-decene exhibited moderate increase in germination efficiency (>5%).
Several individual compounds were able to induce similar plant growth promoting effects at 0.5 µg and 10 ng concentrations. The alkene, 1-decene, promoted Arabidopsis growth at relatively high (0.5 µg) and low (10 ng) concentrations, induced moderate improvement to germination, and caused the greatest increase in plant fresh weight and total chlorophyll content (Figure 6). Of the compounds tested, exposure to 1-decene yielded greatest increase in seed germination, plant fresh shoot weight (39%), and chlorophyll (68%). Solvents isopropyl myristate, (-)-trans-caryophyllen, ocimene, ethanol, and ethanol were tested. These compounds were statistically comparable to untreated control plants. Tomato seedlings were sprayed with 10 ng of 1-decene in ethanol solution had similar fresh weight to control, however, increase in total chlorophyll concentration (10%) (Figure 7 and 8). Preliminary screening using methanol solution appeared to produced greater increase in fresh weight and chlorophyll. Frequency of the compound application and specific concentration should be further examined to determine optimal growing conditions for greenhouse application.
The current research demonstrated the importance of VOCs emitted by Trichoderma species as an important method of fungus-plant interaction and identified specific compounds responsible for plant growth promotion. Exposure to Trichoderma VOCs induced significant changes in Arabidopsis and tomato, promoting all aspects of plant development. Trichoderma are found in diverse environments and readily used in agriculture. This research will bridge the gap in our knowledge about the fungal volatile production in response to frequently changing environmental conditions in modern farming practices. Understanding the impact of nutrient conditions will enable efficient usage of the microorganisms to not only improve crop production but enhance the added benefit of using beneficial fungi. At the practical level, the research will provide the means and tools to accurately and reliably assess potential plant growth promotion to the presence of fungal VOCs. Furthermore, it will help guide future research in discovering the mechanisms behind volatile interaction between fungi and plants. The research will provide new ways to improve farming economics through increased yield and reduced fertilizer use with the aid of these versatile fungi.
Education & Outreach Activities and Participation Summary
The project led to several outreach activities including professional meetings, undergraduate education, and training. I have continued to provide academic support at Rutgers University aimed to focus on increasing the recruitment, academic success and achievement of undergraduate students pursuing agricultural/plant science studies. During the 2014-2015 academic year, I have trained and mentored two Rutgers undergraduate research students and one doctoral student from Iran. Currently, several research publications are being prepared for submission that summarize the results discussed here. I also recently contributed to a book chapter describing Trichoderma and VOC research with an agricultural focus. Research findings were disseminated at several professional meetings including Fungal Genetics, Society of Industrial Microbiology and Biotechnology, and Rutgers. I have given talks at several meetings and my research has been presented by my PI in several international meetings. The results will be publicly available through Rutgers website. I am currently in the process of developing a website to present most of the research discussed here. The website will be available once the research is first available through peer-reviwed papers. I am still in the process of determining the appropriate web host.
Manuscript: Lee, S., Yap, M., Behringer, G., Hung, R., Bennett, J.W. 2016. Volatile organic compounds emitted by Trichoderma species mediate plant growth. Fungal Biology and Biotechnology. 3:7-21.
Book Chapter: Lee, S., Yin, G., Bennett, J.W. 2016. Airborne Signals volatile-mediated communication between plants, fungi, and microorganisms. In: Dighton, J and White, J. (Eds)., The Fungal Community (pp. 521-538). London, England: Taylor & Francis.
Areas needing additional study
Current research can be improved further by cotinuing to assess and determine optimal treatments to be use in greenhouse conditions. Other popular vegetables and fruits grown in greenhouses should also be tested using live fungal cultures and standard compounds. Since the current research measures vegetative responses in plants, additional study should focuse on fruit/seed development.