Evaluating the biological control agent Trichoderma: Enhancement of plant growth and development through biostimulatory volatile treatment

Evaluating the biological control agent Trichoderma: Enhancement of plant growth and development through biostimulatory volatile treatment

Summary

Present research aims to investigate alternative methods of improving the sustainability of greenhouse production by utilizing the biological control agent Trichoderma and its biostimulatory gases.  Previously, we demonstrated that Trichoderma gases (volatile organic compounds [VOCs]) induced growth promoting effects such as increased plant biomass and robust root growth in tomatoes and Arabidopsis.  We began evaluating nutrient conditions to optimize Trichoderma VOC production as well as identify candidate compounds to be used in foliar application.  Of the compounds tested, we identified several that showed significant increase in plant biomass (fresh weight and total chlorophyll concentration).  We also observed differential effects of gases emitted by Trichoderma grown on various fungal substrate.  We also identified solvents that can be safely used on tomatoes for greenhouse application.  Next phase of the research will be to confirm these preliminary findings and to continue developing the method of application for tomatoes.

Objectives/Performance Targets

The proposed research aims to evaluate nutrient conditions to optimize biostimulatory gas production and identify compounds that can be used in foliar application.

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.

Using the split plate bioassay developed previously, we exposed Arabidopsis seeds to Trichoderma grown in fungal media: 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).  From the initial screening, plants responded well to volatiles emitted by Trichoderma grown on OAT and CZA and plants had an increase in fresh weight and total chlorophyll concentration (approximately 30 to 50%).   This experiment will be repeated in January 2015  and fungal medium that induced the most beneficial effects on plants will be selected to be tested on tomatoes.  

b) Evaluate optimized nutrient condition for best stimulation of tomatoes.  
To be completed by spring 2015: use the best fungal medium to evalute tomato response.

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.

Chemical standards were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) to allow for the introduction of defined concentrations.  Using Arabidopsis seeds and vegetative plants, we evaluated 23 compounds:  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. 

Germination assay:   For each chemical treatment, total of 150 seeds were exposed and the experiment was replicated three times (total number of seeds per treatment = 450).  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 had slight increase in germination efficiency (>5%).  

Vegetative exposure assay:  Arabidopsis were surface sterilized and allowed to growth for 14 days prior to start of the exposure.  The plants were exposed to 0.5 ppm of compound for 72 hour.  Total of 20 plants were used per treatment and the experiment was replicated two times (total number of plants per treatment = 40).  Plants exposed to 1-octen-3-ol were significantly smaller in size compare to control.  Compounds such as 1-octen-3-one and nonanal completely killed the plants in 48 to 72 hours.  Plants exposed to octanoic acid, isobutyl alcohol and 1-decene had the largest increase in plant biomass (fresh weight and total chlorophyll concentration).  There was a fresh weight increase of approximately 40% and 44 – 77% in total chlorophyll concentration in plants after gas treatment.  This experiment will be repeated in January 2015 and candidate compounds will be selected to be tested on tomatoes.

b) Expose tomatoes to growth promoting compound(s). To be completed by spring 2015: the plant growth promoting compound(s) will be used to evaluate its effectiveness on tomato growth.

3.  Develop a method of application of biostimulatory volatile compounds in greenhouse setting.
Several solvents were tested on tomato seedlings to evaluate phytotoxicity.  Compounds isopropyl myristate, (-)-trans-caryophyllen, ocimene and ethanol were tested.  These compounds were statistically comparable to untreated control plants.  Once candidate compound(s) is identified and validated, the solvent and fungal compound solution on tomato growth will be assessed.  This experiment will be completed between spring and summer of 2015. 

Accomplishments/Milestones

Brief Timeline of Project Activities:

2014
September: planned experiments, began training undergraduate research assistant, test solvents on tomatoes, began multi media fungal gas production study

October – November: continue to plan and modify experiments, collect and analyze data, screen single compounds (germination and vegetative assays in Arabidopsis)

November:  Invited to present at Fungal Genetics conference

December: Submitted a small short communication paper describing evaluation of nutrients to optimize biostimulatory gas production by Trichoderma, undergraduate student completed a research paper

Work to be completed in 2015:

1) Complete screening fungal media, volatile production experiments and validate on tomatoes
2) Finish screening individual compounds on Arabidopsis, and validate on tomatoes
3) Assess foliar application development using tomatoes
4) Continue mentoring undergraduate students, develop website content, publish manuscripts, and present at 2015 conferences

Project Progression and Commentaries:  Although a lot of work was completed in less than 3 months time, the project progressed slower than expected.  Some of the work was delayed due to severe mechanical/tehcnical issues at the greenhouse and growth chambers. Sudden fluctuation in temperature and humidity lead to very different experimental results and these experiments will be repeated this winter.  Using Arabidopsis to screen numerous compounds and fungal media has proven to be effective and vital to the success of my research.  Finally, increasing the number of compounds to be tested from 12 to 23 was very interesting.  I was able to find several interesting compounds that have never been documented previously to induce growth promotion.  I was also able to solve some of the concentration related phytotoxicity problems. 

Impacts and Contributions/Outcomes

Goals: Provide outreach through professional society meetings, community programs and training in agricultural extension, publications, and undergraduate education

I have been invited to give a seminar at the 28^th Fungal Genetics Conference in Pacific Grove, CA from March 17 to 22, 2015.  I will be presenting my work and obtain feedback from fungal specialists and microbiologists.  This meeting will provide me with the opportunity to further improve the experimental method of volatile production by manipulating fungal growth and metabolism.  I am planning to attend two additional professional society meetings in the summer of 2015.  American Phytopathological Society and Mycological Society of America’s joint meeting with Botanical Society of America will provide me with the opportunity to disseminate my results to plant pathologists, plant biologists, agriculture researchers and much more. I hope to foster collaborative efforts to further improve agricultural sustainability.

I continued to provide academic support at Rutgers.  I mentored and advised undergraduate research students through independent projects.  They learned to present their research through oral and poster presentations.  In 2015, I will continue my efforts to enhance undergraduate research and training. 

By summer of 2015, I plan on creating a website and provide information for people who may not be familiar with Trichoderma. Website will contain comprehensive description of usage of this fungus in agricultural settings.  I will provide literature to discuss the benefits of using these fungi to improve soil quality, improve disease control and resistance, and increase plant growth.  In addition, I will provide a review with recommendations on the usage of various commercial Trichoderma products available as well as the strain used in the proposed research.  I will be publishing manuscripts summarizing my research findings and will be available on the website.

Summary: My research has the potential to determine the most cost effective, sustainable way to utilize the microorganisms in greenhouse vegetable production. Once the research is completed, I will provide recommendations to the industry on the most effective fungal strain (or naturally occurring mixtures) or the most effective compound to apply as a foliar treatment in greenhouse conditions. In addition, I will disseminate data through community outreach, education, and presentation of research to the scientific community. 

Collaborators: