Trichoderma viride produces volatile organic compounds (VOCs) like octanal that inhibit the growth of wood decay fungi such as Neolentinus lepideus. This research aims to determine the impact of T. viride VOCs on N. lepideus protein synthesis. Preliminary results found T. viride VOCs inhibited N. lepideus growth by 29.5% on average and GC-MS identified octanal, a known inhibitory VOC, being produced by T. viride. Future work will isolate individual VOC effects on growth inhibition and analyze protein patterns in N. lepideus exposed to T. viride VOCs using SDS-PAGE.

1. Effect of Trichoderma viride’s Volatile Organic Compounds on Protein Synthesis in
Neolentinus lepideus
Andrew Kocian and Dr. Mary A. Kopecki-Fjetland
Department of Chemistry
St. Edward’s University, 3001 South Congress Ave, Austin, TX 78704
Abstract
Trichoderma viride, a competitive soil fungus, inhibits the growth of wood decaying fungi
such as Neolentinus lepideus via production of volatile organic compounds such as 2-methyl-
1-butanol, heptanal, and octanal. This interaction has the potential to serve as an alternative
to chemical treatment of fungal wood infections. The literature reports that fungal VOCs can
cause a decrease in the synthesis of specific proteins. This research aims to determine the
impact of T. viride’s VOCs on protein synthesis in N. lepideus furthering the understanding of
its potential biocontrol nature. SPME and GC-MS analysis confirm the production of T. viride
VOCs, specifically identifying octanal. Other preliminary studies confirm N. lepideus exhibits
growth inhibition when exposed to the VOCs of T. viride. Future work includes further
isolation of total protein from N. lepideus grown in the presence and absence of T. viride
followed by analysis of varying protein patterns using SDS PAGE over different times.
Additionally, the degree of N. lepideus inhibition by individual T. viride VOCs will be
examined.
Background
Microbial volatile organic compounds (MVOCs or VOCs), bacterial or fungal, have been
extensively documented to interact with soil ecosystems (Ortiz-Castro 2009). An example is
the boost in root growth of Arabidopsis thaliana when it shares an atmosphere with T. viride
(Piechulla et al 2015). T. viride inhibits the growth of wood decaying fungi such as N. lepideus
via production of volatile organic compounds such as 2-methyl-1-butanol, heptanal, and
octanal. This interaction has the potential to serve as an alternative to chemical treatment of
fungal wood infections. Since fumigation of wooden structures with conventional fungicides
presents a human health risk, production of fungitoxic concentrations of VOCs by T. viride in
situ could remove wood pathogenic species without damage to the structure, environment,
or humans. A similar mechanism has been shown in the protection of plant species against
plant pathogens (Piechulla et al 2015).
The literature reports that fungal VOCs can cause a decrease in the synthesis of specific
proteins in fungal targets. Wheatley showed that in Serpula lacrymans that was exposed to
the VOCs of T. viride, certain proteins were not synthesized after several days when
compared to S. lacrymans that was not exposed to VOCs (2002). The loss of these proteins
coincided with the reduction of growth of S. lacrymans that was exposed to the VOCs.
Additionally, Wheatley found that the growth of Postia placenta, Gloeophyllum trabeum, and
Trametes versicolor was inhibited by the VOCs of T. viride (1997). These results raise the
question if T. viride inhibits growth of target fungi by inhibiting certain proteins contingent on
full growth and development of the target. Answering this question gives a hint into the
means of how T. viride inhibits the growth of its targets. Therefore, this research aims to
discover the effect T. viride’s VOCs has on the protein synthesis of N. lepideus.
Methods
(Wheatley 2002)
Figure 1: Protein
patterns affected by
VOCs.
Expose N.
lepideus
Extract N.
lepideus
Protein
SDS PAGE
Confirm
Growth
Inhibition with
Strain
Measure
Growth
Inhibition
Detect T.
viride’s VOCs
via GC-MS
N.
lepideus
T. viride
Parafilm
seal
Results
Figure 2: A) Inhibition of growth and production of VOCs were confirmed. Strains ATCC 28038 and ATCC
12653 were used as models. Inhibitory behavior, as well as VOC production were verified initially. VOCs
were collected via headspace-solid phase microextraction and analyzed using GC-MS. B) N. lepideus and T.
viride were grown together via atmospheric connection. Petri dishes of each organism were inverted over
each other and sealed aseptically with parafilm.
A) B)
Set
Percent
Inhibition
Trial 1 30%
Trial 2 29%
Average 29.5%
Figure 3: A) Growth inhibition of N.
lepideus by T. viride. N. lepideus was
exposed to T. viride VOCs for eight days.
Values for each trial represent an average
of three samples. B) The percent inhibition
of growth calculated for each trial.
A)
B)
Acknowledgments
This work was supported by the Welch Foundation and the Department of Chemistry in the School of Natural Sciences
at St. Edward’s University.
References
Humphris, S. N., Wheatley, R. E., and Bruce, A. (2001) The Effects
of Specific Volatile Organic Compounds Produced by Trichoderma
Spp. on the Growth of Wood Decay Basidiomycetes.
Holzforschung. 10.1515/hf.2001.038
Humphris, S. N., Bruce, A., Buultjens, E., and Wheatley, R. E.
(2002) The effects of volatile microbial secondary metabolites on
protein synthesis in Serpula lacrymans. FEMS Microbiology
Letters. 210, 215–219
Kottb, M., Gigolashvili, T., Großkinsky, D. K., and Piechulla, B.
(2015) Trichoderma volatiles effecting Arabidopsis: from
inhibition to protection against phytopathogenic fungi. Front.
Microbiol. Frontiers in Microbiology. 10.3389/fmicb.2015.00995
Lee, S., Yap, M., Behringer, G., Hung, R., and Bennett, J. W. (2016)
Volatile organic compounds emitted by Trichoderma species
mediate plant growth. Fungal Biology and Biotechnology.
10.1186/s40694-016-0025-7
Parizi, T. E., Ansari, M., and Elaminejad, T. (2012) Evaluation of the
potential of Trichoderma viride in the control of fungal pathogens
of Roselle (Hibiscus sabdariffa L.) in vitro. Microbial Pathogenesis.
52, 201–205
Wheatley, R., Hackett, C., Bruce, A., and Kundzewicz, A. (1997)
Effect of substrate composition on production of volatile organic
compounds from Trichoderma spp. Inhibitory to wood decay
fungi. International Biodeterioration & Biodegradation. 39, 199–
205
Figure 4: Inhibition of N. lepideus by T.
viride. Row one with red arrows
represents N. lepideus control. Rows 2
and 3 represent N. lepideus challenged by
VOCs emitted from actively growing T.
viride.
Figure 5: A) Mass spectrum of
the pure octanal peak shown in
B (upper spectrum), and mass
spectrum of octanal peak in T.
viride VOC sample (lower
spectrum). B) Chromatogram of
T. viride VOCs with an identified
octanal peak. C) Chromatograms
of standards that have been
identified in the literature to be
inhibitory to wood decaying
fungi. The octanal standard peak
matches the peak of T. viride’s
headspace sample. These
preliminary results indicate that
strain ATCC 28038 is producing
an array of VOCs.
Octanal
2-Methyl-1-Butanol
Heptanal
C)
39.0
55.5
50.6
71.6
0
10
20
30
40
50
60
70
80
Growthin8days(mm)
Trial 1
Growth inhibition of N. lepideus by T. viride
Trial 2
Exposed Not Exposed
Discussion
Growth inhibition studies indicate that T. viride inhibits the growth of N.
lepideus by almost 30%. This average value is slightly lower than the values
reported in the literature, but it should be noted that percent inhibition is
dependent on variables such as ambient temperature, isolation location,
growth media, and target fungi of T. viride.
GC-MS analysis indicates that T. viride is producing the VOC, octanal, one
of the aldehydes previously identified in the literature to cause inhibition in
wood decaying fungi. Preliminary extraction and separation of proteins
utilizing SDS-PAGE have proven inconclusive regarding varying protein
production in N. lepideus exposed to T. viride vs the control.
Future work includes utilization of various SPME conditions to enhance
the concentration of fungal VOCs isolated, propagation of N. lepideus in the
presence of individual VOCs in order to determine more specifically the
impact on fungal growth inhibition and SDS PAGE analysis of N. lepideus
protein when the organism is exposed to actively growing T. viride.
B)
A)