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Natural Antimicrobial peptides in Agriculture
NATURAL ANTIMICROBIAL
PEPTIDES AS GREEN
MICROBICIDES IN AGRICULTURE
A PROOF OF CONCEPT STUDY ON THE TYROCIDINES FROM SOIL BACTERIA
Marina Rautenbach, Arnold Johann
Vosloo, Wilma van Rensburg and Yolanda Engelbrecht
30 DECEMBER 2015
Natural Antimicrobial peptides in Agriculture
NATURAL ANTIMICROBIAL PEPTIDES AS GREEN MICROBICIDES IN AGRICULTURE
Natural Antimicrobial peptides in Agriculture
This Research Report was prepared under the Research Funding Programme, ‘Research and Policy Development to
Advance a Green Economy in South Africa'
By:
Marina Rautenbach, PhD
Head of BIOPEP Peptide Group
Professor of Biochemistry
Department of Biochemistry
Stellenbosch University
Private Bag X1, Matieland 7602
Stellenbosch, South Africa
Email: [email protected]
Tel: +27-218085872/8
Fax: +27-218085863
UNIVERSITEIT • STELLENBOSCH • UNIVERSITY
jou kennisvennoot • your knowledge partner
Natural Antimicrobial peptides in Agriculture
GREEN FUND
RESEARCH AND POLICY DEVELOPMENT TO ADVA NCE A GREEN ECONOMY IN SOUTH AFRICA
GREEN ECONOMY RESEA RCH REPORTS
The Government of South Africa, through the Department of Environmental Affairs, has set up the Green Fund to
support the transition to a low-carbon, resource-efficient and pro-employment development path. The Green Fund
supports green economy initiatives, including research, which could advance South Africa’s green economy transition.
In February 2013, the Green Fund released a request for proposals (RFP), ' Research and Policy Development to Advance
a Green Economy in South Africa’, inviting interested parties with relevant green economy research projects to apply
for research funding support. The RFP sought to strengthen the science-policy interface on the green economy by
providing an opportunity for researchers in the public and private sectors to conduct research which would support
green economy policy and practice in South Africa. Sixteen research and policy development grants were awarded in
2013. This peer-reviewed research report series presents the findings and policy messages emerging from the research
projects.
The Green Economy Research Reports do not represent the official view of the Green Fund, Department of
Environmental Affairs or the Development Bank of Southern Africa (DBSA). Opinions expressed and conclusions arrived
at, are those of the author/s.
Comments on Green Economy Research Reports are welcomed, and may be sent to: Green Fund, Development Bank
of Southern Africa, 1258 Lever Road, Headway Hill and Midland 1685 or by email to [email protected].
Green Economy Research Reports are published on:
www.sagreenfund.org.za/research
Please cite this report as:
Rautenbach, M., Vosloo J.A., Van Rensburg, W. and Engelbrecht, Y., 2015, Natural antimicrobial peptides as green microbicides in agriculture: A proof of concept study on the tyrocidines from soil bacteria, Green Economy Research Report, Green Fund, Development Bank of Southern Africa, Midrand.
Natural Antimicrobial peptides in Agriculture
TABLE OF CONTENTS
EXECUTIVE SUMMARY ............................................................................................................................................................. 7
RESEARCH TEAM ............................................................................................................................................................... 8
ABBREVIATIONS ................................................................................................................................................................ 9
LIST OF FIGURES .............................................................................................................................................................. 11
LIST OF TABLES ................................................................................................................................................................ 13
1. INTRODUCTION ........................................................................................................................................................... 14
2. BACKROUND TO RESEARCH / CONTEXT / PROBLEM STATEMENT ............................................................................... 14
3. AIMS AND OBJECTIVES / RESEARCH QUESTIONS ........................................................................................................ 15
Phase I ......................................................................................................................................................................... 15
Objectives 1-4 .......................................................................................................................................................... 15
Milestones 1-3 ......................................................................................................................................................... 15
Phase II ........................................................................................................................................................................ 16
Objectives 5-10 ........................................................................................................................................................ 16
Milestones 4-9 ......................................................................................................................................................... 16
4. LITERATURE REVIEW ................................................................................................................................................... 16
5. METHODOLOGY .......................................................................................................................................................... 20
Phase I ............................................................................................................................................................................. 20
Research Purpose and Design ................................................................................................................................. 20
Design and optimising fermentation of producer cultures ...................................................................................... 21
Up-scaling and optimising purification for formulation and field trials ................................................................... 21
Tailoring natural peptide microbicide combinations ............................................................................................... 21
Development and testing of selected antifungal peptide formulations .................................................................. 21
Phase II ............................................................................................................................................................................ 21
Research Purpose and Design ................................................................................................................................. 21
In vivo evaluation of peptide microbicide formulation(s) toxicity on insects ........................................................... 21
In vivo evaluation of peptide formulation on the selected vase flowers ................................................................. 22
In vitro assessment of the surface activity of peptide microbicide formulations .................................................... 22
In vivo evaluation of peptide microbicide formulation on seasonal fruits ............................................................... 22
In vivo evaluation of peptide formulations in selected plant cultures and micro-propagated plants ....................... 22
In vivo evaluation of peptide microbicide formulation on woody plants ................................................................. 23
6. CHALLENGES AND CONSTRAINTS ............................................................................................................................... 23
7. RESULTS/FINDINGS...................................................................................................................................................... 24
Phase I Research Results ............................................................................................................................................. 24
Design and optimising fermentation of producer cultures ...................................................................................... 24
Up-scaling and optimising economical purification ................................................................................................. 24
Tailoring natural peptide microbicide combinations ............................................................................................... 27
Development and testing of formulations of the selected antifungal peptides and combinations .......................... 28
Natural Antimicrobial peptides in Agriculture
Phase II Research results ............................................................................................................................................. 30
In vivo evaluation of peptide microbicide formulation toxicity on bees and nematodes........................................ 30
In vivo evaluation of peptide microbicide formulations on selected cut flowers .................................................... 32
In vitro assessment of the surface activity of peptide microbicide formulation ..................................................... 34
In vivo evaluation of peptide microbicide formulations on selected seasonal fruits .............................................. 37
In vivo evaluation of peptide microbicide formulations in plant cultures ............................................................... 38
In vivo evaluation of peptide microbicide formulation on woody plant grafts and cuttings ................................... 41
8. CONCLUSIONS ............................................................................................................................................................. 44
8.1 General conclusions .............................................................................................................................................. 44
Advantages of our antimicrobial peptides and formulations .................................................................................. 44
Impact on Agriculture .............................................................................................................................................. 44
Impact on Industry .................................................................................................................................................. 44
8.2 Key Policy Messages .............................................................................................................................................. 45
Improving public knowledge, attitudes, skills, and abilities .................................................................................... 45
Changing practices, decision making, policies (including regulatory policies), social actions ................................. 45
Improving social, economic, civic, or environmental conditions ............................................................................. 45
8.3 Recommendations for Further Research / Action ................................................................................................. 45
AKNOWLEDGEMENTS ..................................................................................................................................................... 46
REFERENCES .................................................................................................................................................................... 47
ANNEXURE A ................................................................................................................................................................... 51
Broad spectrum antibacterial activity of the TRCs and TCN against Gram-positive bacterial strains.......................... 51
Antibacterial activity of the TRCs and TCN against Gram-negative bacterial strains ................................................... 52
Broad spectrum antifungal activity of the TRCs and TCN against fungal pathogens ................................................... 53
Natural Antimicrobial peptides in Agriculture
EXECUTIVE SUMMARY
The global food security is threatened throughout the production chain. From the farmer to the consumer losses via
weak planting material and microbial infections have both a major economic impact and health implications.
Antimicrobial peptides (AMPs) are natural bio-control agents, which are part of the first line of defence of living
organisms. Certain antimicrobial peptides have also phytostimulatory activity, related to induced systemic resistance
(ISR) in plants. They are Nature’s weapon of choice in maintaining a natural microbial ecology and therefore
exceptional candidates for eco-friendly microbicides and phytoactives. However, AMPs are not utilised in agriculture
and there is very little research on the application of antimicrobial peptides in agriculture. There is thus a need to
improve the knowledge in this field, with a particular emphasis on natural peptides produced by soil organisms.
In this project we aim to translate basic research into applications by specifically targeted food security problems in
selected steps in the production chain with natural peptide antimicrobials and phytostimulants produced by beneficial
soil bacteria. First, we aimed to prevent plant diseases through the production of high quality, pathogen free plant
material by utilising our natural antimicrobial and phytostimulatory peptide products form selected producer organisms
in plant cultures. To target this pre-harvest section of the food production chain we focussed on cyclic peptides
produces by a soil bacterium, namely the tyrocidines and analogues that are known to have potent antifungal and
antibacterial activity. These cyclic peptides are highly stable and would be able to withstand biological exposure, but
are still fully biodegradable to nutrients. Second, the selected peptides were utilised in antimicrobial formulations and
materials to improve sterilisation, germination and plant propagation.
Good progress in this extensive translational research project was made over the 20 months of the project and we
reached the majority of objectives and milestones. The good progress entailed first the successful production and basic
formulation of tailored antimicrobial peptide mixtures. Second, these natural antimicrobial peptide formulations were
successfully applied in several field trials at two nurseries, as well as in controlled laboratory trials. The key findings in this
study are (1) that we are able to economically produce antimicrobial peptides, (2) that the natural peptides have in
vivo activity with potential plant yield improvement under nursery conditions and (3) that we are able to create robust
antimicrobial materials. These antimicrobial materials are being developed further for packaging, filtration and general
sterilisation. However, more research is needed on the medium and large scale production, as well as the bio-stability of
our peptide formulations. The study on plant cultures and micro-propagation will be extended, focusing on the
influence of our peptide formulations on phytostimulation, ISR and soil fertility.
With the movement against harmful unnatural chemicals in agriculture, our interactions with nurseries confirmed the
need for alternative green products in the market. Our collaboration on the nursery trials and feedback indicated that
nurseries are willing to use green alternatives with potential to replace harmful chemicals, which is highly encouraging
for an attitude change in favour of natural product utilisation in agriculture. We remain focussed on engaging key stake
holders, such as nurseries in order to promote the use of natural products, rather than synthetic chemicals that is
potentially harmful to our health and the environment. The positive results obtained in this exploratory project
demonstrated the potential of the natural antimicrobial peptides in agricultural and industrial applications, and may for
the first time offer a viable natural alternative to nurseries. Our aim to develop and promote our peptide products only
for the controlled use of low amounts natural peptides in nurseries for plant propagation, via micro-propagation, grafts
and plant cultures. If nurseries can then provide the farmer with healthy more resistant plants, it will indirectly lower the
need for chemical biocides.
Ms R Valashiya Intern
Green Fund Grant
Ms H Barkhuizen PhD Student
Natural Antimicrobial peptides in Agriculture
RESEARCH TEAM
Table 1: Summary of the Green Fund Grant collaborators and team members who contributed to the success of this
project over the 20 month duration the project.
Name Position Role Funded by Period
Prof M Rautenbach Principle investigator
Project coordination and leadership, reporting, training
Stellenbosch University Permanent staff member
Prof JL Snoep Collaborator Collaboration on production via fermentatio and on peptide production models
n Stellenbosch University Permanent staff member
Dr P Hills
Prof M Vivier
Dr L Hofmann
Collaborator
Collaborator
Collaborator
Collaboration on micro-propagation study
Collaboration on grape plant culture study
Collaboration on cut-flower trials.
Stellenbosch University
Stellenbosch University
Stellenbosch University
Permanent staff member
Permanent staff member
Permanent staff member
Dr M Lutz Collaborator Collaboration on polymers and membranes
used for solid phase activity study Stellenbosch University
Permanent staff member
Dr M Stander Collaborator Mass spectrometric analysis of peptide extracts for quality control.
Stellenbosch University Permanent staff member
ARC Agricultural Dr M Allsopp Collaborator Collaboration on bee toxicity studies
Research Council Plant Protection Research
Permanent staff member
Dr H Beims Collaborator Collaboration on bee larvae toxicity and bee pathogen studies
Institute Technische Universität
Braunschweig (Germany) Permanent staff member
Mr R Joubert Collaborator Collaboration on grape grafting trials Fleury Nursery Owner
Mr J Heyns Collaborator Collaboration on grape grafting trials Fleury Nursery Permanent staff member
Mr M Prinsloo Collaborator Collaboration on grape grafting trials Stargrow Permanent staff member
Mr E Burger Collaborator Collaboration on apple grafting trials Stargrow Permanent staff member
Mr P Louw Collaborator Collaboration on blueberry trials Rosenhof Nursery Permanent staff member
Mr W van Rijswik Collaborator Collaboration on peaches grafting trials Rosenhof Nursery Permanent staff member Co-design, set-up and management of
Dr N Lombard Production manager
production/ purification unit. Optimization of medium scale production and purification.
Green Fund Grant 1 Apr 2014 –
30 Sept 2015
Dr Y Engelbrecht Field trial
manager
Ms WJ Bredell Laboratory
Manager
Coordinator of agricultural field trials. Green Fund Grant 1 Oct 2014 - 30 Sept 2015
(part time)
General laboratory management. Financial
management of Green Fund. Stellenbosch University Permanent staff member
Medium scale production and purification of
tyrocidines Green Fund Grant 1 July - 31 Dec 2014
Mr WW Adams Intern Medium scale production and purification of tyrocidines Plant culture studies and trials with cyclic
15 Feb - 30 Nov 2015
Dr AM Troskie Post-doctoral fellow
Mnr JA Vosloo PhD student
peptides. Cut flower trials. Worm toxicity study.
Optimizing of production and purification of peptides. Tailoring and formulation of peptides. Bee toxicity study.
NRF postdoctoral fellowship
NRF bursary Green Fund Grant
1 Apr 2014 – 30 March 2015
Jan 2012-Dec 2014
1 Jan - 30 Sept 2015
Plant culture studies and trials with natural
plant peptides NRF bursary 1 Jan 2013- 30 Dec 2015
Ms W van
Rensburg MSc Student Solid phase antimicrobial activity studies
Mr WE Laubscher MSc Student Identification, production, purification and testing of natural antimicrobial peptides
Mr S Berge MSc Student Tailored bacterial production, purification and testing of cyclic antimicrobial peptides
NRF Bursary
Green Fund
Harry Crossley Trust Bursary
Green Fund Grant
NRF CSUR bursary
1 Jan 2013 - 30 Dec 2014
1 Jan - 30 Nov 2015
1 Jan 2015 - 30 Dec 2015
1 Jan - 30 Nov 2015
1 Feb- 30 Sept 2015
1 Feb 2015 - 30 Dec 2016
Ms M Barnard Hons student Production of tailored peptide mixtures NRF Bursary 1 Jan - 31 Dec 2014
Ms D van Rooyen Hons Student Testing of natural antimicrobial peptides NRF bursary 1 Jan - 31 Dec 2014
Natural Antimicrobial peptides in Agriculture
ABBREVIATIONS ACN acetonitrile
Asn asparagine
AMP(s) antimicrobial peptide(s)
Cellulose CL
Cellulose acetate CA
Chitin spun on cellulose whiskers CH-CL
Chitin spun on PMM fibres CH-PMM
Comm commercial biocide
DiM dimethoate
DMF dimethylformamide
DLS dynamic light scattering
EDTA ethylenediaminetetraacetic acid
ESMS electrospray mass spectrometry
EtOH ethanol
EU European Union
Gln glutamine
g gram
Glc glucose
GRAS generally recognised as safe
GRM(s) linear gramicidin(s)
GS gramicidin S
GM genetically manipulated
High density cellulose HDC
HPLC high performance liquid chromatography
HCl hydrochloric acid (“pool acid”)
Ile isoleusine
ISR induced systemic response
kg kilogram
L litre
LCN A leucocin A (bacteriocin, AMP)
Leu leucine
Lys lysine
MIC minimum inhibitory concentration
mixed cellulose ester NCCA
mL millilitre
mg milligram
MS mass spectrometry
m/v mass per volume mixture
NaCl sodium chloride (“table salt”)
NaOCl2 sodium hypochlorite (“bleach”)
NDA non-disclosure agreement
Orn ornithine
Phe phenylalanine
Polycarbonate PC
Poly(methyl methacrylate) PMM
Polypropylene PP
Polystyrene PS
Polyvinylidene difluoride PVDF
Pro proline
QSAR qualitative structure-activity relationship
SEM standard error of the mean
spp species in plural
Suc sucrose
TCN tyrothricin
Natural Antimicrobial peptides in Agriculture
Trp tryptophan
TrcA tyrocidine A
TrcB tyrocidine B
TrcC1 tyrocidine C
TRC(s) tyrocidine(s)
Tyr tyrosine
UPLC-MS ultra-performance liquid chromatography linked to mass spectrometry
g microgram
Val valine
v/v volume per volume mixture
Natural Antimicrobial peptides in Agriculture
LIST OF FIGURES
Figure 1: The chemical structure of tyrocidine A, one of the major TRCs. Conventional three-letter abbreviations are
used for amino acid residues, except Orn for ornithine. The alternative amino acid residues for the other
peptides in the TRC complex are indicated at positions 3, 4, 7 and 9. Lys in position 9 leads to A1, B1 and C1
analogues. Phe in positions 3 and 4 leads to the A, A1 analogues, Trp in position 3 to the B, B1 analogues and
Trp in 3 and 4 position to the C, C1 analogues. Tyr in position 7 leads to the tyrocidines, Trp to the
tryptocidines and Phe to the phenycidines.
Figure 2: The strategy outline of the approach followed in project on “Natural antimicrobial peptides as green
microbicides in agriculture: A proof of concept study on the tyrocidines from soil bacteria”.
Figure 3: Flow diagram of the production and purification steps used in the optimised production of TCN and TRC
preparation.
Figure 4: A graphic depiction of the predicted cost per gram of the three different peptide preparations utilising our
optimised production and purification protocol. Calculations are based on the data given in Table 3 and
exclude labour, laboratory space hire and general overheads.
Figure 5: Isobolograms of the combined activity of TrcA and TrcB against the fungal target A. fumigates, and TrcC
and TrcB against the bacterial target Bacillus subtilis. Fractional inhibition concentrations (FICs) falling below
the red line in the green triangle indicate synergistic activity. Each data point is the mean of 4
determinations with error bars representing SEM.
Figure 6: Comparison of the relative activity of TCN75 toward the representative fungal pathogen, Aspergillus
fumigatus and Gram-positive bacterium, Bacillus subtilis. TNC75 was dissolved in either 1.5% (v/v) ethanol
(EtOH) or 1.0% (v/v) DMF alone or in the presence of 5% (m/v) of either sucrose (Suc) or glucose (Glc).
Inhibition parameters determined in EtOH was set as 100%. Statistical analysis was done using Bonferroni’s
Multiple comparison test (One Way ANOVA) with P<0.001 when comparing formulations (with and without
sugars) in the two solvents and P<0.01 when comparing the sugar formulations relative to the respective
solvents alone. Each data point is the mean of 3-50 determinations with error bars representing the SEM.
Figure 7: Consumption of TCN75DS by adult African honey bees relative to the 1% DMF/50% Suc, and DiM controls. A.
Amount of TCN75DS in µg consumed per bee in each of the respective TCN75DS feeding solutions. B. The
relative percentage mortality of bees observed in each of the different feeding solutions over the 72 hour
feeding period corrected relative to the natural mortality in the 50% Suc group using Abbots correction
(Abbot 1925). Statistical analysis was done using Bonferroni’s Multiple comparison test (Two Way ANOVA)
with *P<0.001 relative to 1% DMF/50% Suc. Each data point is the mean of 4 determinations with error bars
representing the SEM.
Figure 8: Comparison of the retrieval of Apis meliffera mature honey bees fed with either control (1% DMF/50% Suc) or
1500 mg/L TCN75DS feeding solutions for 2 days and then returned to their hives of origin. With A showing the
average % retrieval compared to the control and B the retrieval in the respective hives compared to the
untreated controls. Each data point is the mean of 4 determinations with error bars representing the SEM.
Figure 9: The relative percentage mortality of Apis meliffera honey bee larvae after a single exposure to a range of
concentrations of TCN75DS together with the insecticide DiM at day 4. Statistical analysis was done using
Bonferroni’s Multiple comparison test (One Way ANOVA). The relative mortality after exposure to the vehicle
containing 0.8% DMF was compared to each of the respective treatments at days 5, 6 and 7 # P<0.001; $
P<0.01; * P<0.05. Each data point is the mean of 9 determinations with error bars representing the SEM.
Figure 10: Effect of vase water treatment on the water uptake of Blue larkspur (Delphinium hybrid) and the African
daisy (Gerbera spp) on the number of usable/saleable flowers over time. Controls contained only tap water,
those with commercial product received the dosage as specified by supplier.
Figure 11: Photographic evidence of the influence of the different additives to the vase water of Gerbera mermaid
(two panels on left) and Gerbera larreia (two panels on right) at 12 and 16 days.
Figure 12: Comparison of retained antimicrobial activity of different materials treated with TCN75 F4. The inhibitory
activity was determined in a low nutrient environment with high bacterial cell count (7x104 Micrococcus
luteus cells per well or 5 mm filter disk) using the Alamar Blue viability assay. Bars represent the average of 6-9
determinations with SEM.
Natural Antimicrobial peptides in Agriculture
Figure 13: The effect of washing of CL filters treated with TCN75 F4 with different solvents on the sterility of the filters as
determined with a vitality assay with Micrococcus luteus as bacterial contaminant (A). Each data point
represents the mean of at least 24 determinations with SEM. The graph on B shows the retention of activity
over time on the antibacterial activity of TCN75 F4 treated CL. Each data point represents the mean of 6-30
determinations with SEM. Statistical analyses in A and B were done using Bonferroni’s Multiple comparison
test (One Way ANOVA) with *** P<0.001. Bars represent the average of 6-9 determinations with SEM.
Figure 14: The effect of CL filters treated at varying concentrations of TCN75 (0, 5, 25 and 50 mg/L) on root length (A)
and total biomass of tomato seeds after germination (B) on the filters. For each filter treatment the
germinated plants of 75-100 seeds were analysed. Statistical analysis was done using Bonferroni’s Multiple
comparison test (One Way ANOVA) with control compared to TNC75 treatments with ** P<0.01; *** P<0.001.
Bars represent the average of 40-95 determinations with SEM.
Figure 15: The influence of TRC85 F1 on the vitality and growth of Vitis vinifera (grapevine) cuttings over two months.
The bar graph shows the comparison of TRC85 F1 supplementation with control media of growth parameters
over two months, with photographic evidence on a selection of cultivars after two months. Statistical analysis
was done using Bonferroni’s Multiple comparison test (One Way ANOVA) with control compared to TRC85 F1
treatment with * P<0.0%; ** P<0.01; *** P<0.001. Bars represent the average of 12 determinations with SEM.
Figure 16: The influence of TNC75 F4 on the vitality and growth of Arabidopsis thaliana micro-propagated seedlings
over 5 weeks. The bar graph shows the comparison with control media of growth parameters, with
photographic evidence on a selection of cultivars after two months. Statistical analysis was done using
Bonferroni’s Multiple comparison test (One Way ANOVA) with control compared to TNC75 F4 treatment at
with * P<0.05. Bars represent the average of 16 determinations with SEM.
Figure 17: The process of grape vine grafting at Fleury Nursery in Wellington with grafting process of the grape cultivars
to a robust root cultivar performed by skilled artisans (A); waxed grape vine grafts after treatment in wood
pallets (B); grafts covered with wood savings for the 1-2 months incubation period (C); counting of young
geminated grape vine plants in vineyard after 3 months (D); harvesting of grape vine plants after 10-11
months €; sorting and grading of young vines (F); storage of viable young plants (G) and grape vine plant
bundles ready for delivery to farmers (H).
Figure 18: The influence of TNC40A50 on the germination and growth of Vitis vinifera (grapevine) grafts after three
months and the harvested Class 1 yields. The bar graph shows the comparison with control treatments. The
number of grafts in each trial is indicated above each bar. The photographic evidence of the germinated
plants in the vineyard is shown after two months. Statistical analysis was done using Students test with control
compared to TNC40A F4 treatment with ** P<0.01.
Natural Antimicrobial peptides in Agriculture
LIST OF TABLES
Table 1: Summary of the Green Fund Grant collaborators, advisors and team members who contributed to the
success of this project over the 20 month duration of the project.
Table 2: Summary of the average tyrothricin yield from cultures of Brevibacillus parabrevis grown in different media.
Table 3: Summary of tyrothricin production costs to produce 500 g of crude tyrothricin preparation or either of the
two purified preparations. The costing excludes labour, laboratory space costs, general overhead costs, but
includes electricity and instrumentation costs.
Table 4: Summary of the chemical composition of TNC75 preparation as determined with UPLC-MS. Refer to Fig. 1 for
more detail on the chemistry of the tyrocidines and analogues.
Table 5: Influence of additive in TRC formulation on the peptide activity towards the target cell. The peptides were
pre-incubated for 60 minutes in NaCl, KCl, MgCl2 or CaCl2 at concentrations ranging from 2.0-100 mM salt.
Table 6: Summary of the TCN and TRC formulations and their applications in this study.
Table 7: Summary of the characteristics and conventional use of selected filters utilised in this study and the influence
of treatment with TCN75 F4.
Table 8: The “in vivo” antifungal activity of TCN75 in the presence and absence of grapevine stalk material as
determined with a viability assay. The percentage viable spores compared to the control are indicated.
Table 9: Summary of orange treatment (sanitation/protection) trial with natural antimicrobial peptide preparations.
Table 10: Summary of completed and ongoing nursery trials in the Western Cape.
Natural Antimicrobial peptides in Agriculture
1. INTRODUCTION
Pre- and post-harvest production losses in the agricultural sector, due to microbial disease, are serious problems which
severely impacts productivity and global food security. With the existing chemical antimicrobial agents losing potency
and increased global opposition to their use, there is a need for natural bio-control agents. A large group of natural bio-
control/microbicide agents already exists, namely the antimicrobial peptides (AMPs) that are part of the first line of
defence of all living organisms. Our group have identified numerous highly potent natural AMPs with potential as green
microbicides, in particular the tyrocidines (TRCs) and analogues from the soil bacterium Brevibacillus parabrevis (also
known as Bacillus aneurinolyticus and Bacillus Brevis). This project entailed a proof of concept study to utilise the TRCs
and analogues as green/biodegradable microbicides to (1) prevent latent fungal pathogen carry-over into plant
cultures/nursery propagated plants and (2) afford protection against post-harvest fungal pathogens infecting harvested
produce. We first focussed on biotechnological up-scaling of our current small scale production/isolation systems and
then on formulation of the TRCs for selected agricultural and biotechnological applications. Second, we used the
produced and formulated natural peptide microbicides in proof of concept trials in selected applications, namely as
sterilising agents in plant cultures, micro-propagation, plant grafting, fruit protection and to prolong the vase life of cut
flowers, as well as in materials that can potentially be used as packaging materials. The natural peptide microbicides
are aimed to protect plants from the seed, graft, cutting or tissue culture phase through to the established young plants
in nurseries and final products in the market. The positive results of this project that are reported here can potentially
lead to green microbicides that could support organic/natural farming practices and contribute to sustainable
production of crops and therefore become part of the Green Economy in South Africa.
2. BACKROUND TO RESEARCH / CONTEXT / PROBLEM STATEMENT
In today’s competitive fresh produce market success is driven by product quality and perceived wholesomeness by the
health conscious consumer. Producers can no longer afford to supply inferior quality produce to the modern consumer
that demands products produced with a minimal impact on the environment. The greatest threat to the fresh produce
market still remains the reduction in yield and quality due to microbial infections, especially fungal pathogens. Pre-
harvest infection by microbial pathogens results in a loss of approximately 16% in global food production each year,
with a further loss of up to 50% as a consequence of post-harvest infections, particularly in developing countries, which
has the potential of threatening global food security (Chakraborty & Newton 2011; Montesinos & Bardaji 2008).
Traditionally plant diseases have been treated with chemical microbicides, but with the development of resistance and
pressure by the consumer to reduce the dependence on chemical microbicides producers are faced with a greater
challenge of protecting their valuable commodities (Vidaver 2002). Also, control measures during storage and transport
of harvested products are frequently failing and this serious problem must be addressed in the light of a growing
consumer resistance to foreign chemicals in food and beverages.
With the conventional antifungal agents failing against many of the fungal pathogens, the increased global resistance
to the use of fungicides and movement towards more natural/organic farming practice, in particular in the European
Union (EU) (Williams 2011), there is an urgent need for natural and safe bio-control agents. Alternatives to chemical
microbicides such as microbial bio-control agents have given producers an environmentally friendly means of
protecting their produce. However, an ever changing agricultural environment and poorly controlled application of a
variety of commercial microorganisms of which some produce small proteins namely antimicrobial peptides (AMPs) and
some have antagonistic action towards another. Failure due to antagonism and loss of microbial viability has led to the
impression that the efficacy of microbial bio-control is unpredictable and there is no guarantee of protection against all
the causative pathogens. Our approach is to target other areas of the production chain, particularly the prevention of
diseases in new orchards and vineyards through the planting of high quality, pathogen free plant material. Quality of
the new planting material supplied by nurseries, however, is often suspected by farmers when they lose hectares of
young plants because of single organism infections.
The direct use of AMPs, rather than their bacterial producers may be a solution to the biocide resistance and
environmental problem. AMPs are natural bio-control agents, which are part of the first line of defence of living
organisms. AMPs are found throughout the prokaryotic and eukaryotic kingdoms and show a broad range of activity
toward Gram-positive and Gram-negative bacteria, fungi and viruses (Jenssen, Hamill, & Hancock 2006). They are
Nature’s weapon of choice in maintaining a natural microbial ecology and therefore exceptional candidates for eco-
friendly microbicides. Many of the beneficial AMPs are produced naturally in the healthy soil and water bodies by
bacteria and are natural products that are biodegraded to nutrients. Plants also produce a host of AMPs as a defence
mechanism against pathogens (Broekaert et al. 1995, Lay & Anderson 2005). AMPs are therefore an alternate
underexplored class of antimicrobial agents which have a novel mechanism of action and alternate cellular targets
compared to conventional antibiotics and biocides (Jenssen et al. 2006; Rautenbach & Hastings 1999, Sang & Blecha
2009).
Natural Antimicrobial peptides in Agriculture
The AMP research groups at University of Stellenbosch identified a number of antimicrobial peptide candidates,
including a group of highly potent cyclic peptides, the tyrocidines (TRCs), the analogous gramicidin S (GS), selected
plant defensins and a number of lipopeptides. The development and production of natural microbicides with
antimicrobial peptides as the active ingredients in South Africa could be strategically important not only for South
African agriculture, but also that of Africa. We therefore opted to focus on the production of antifungal cyclic AMPs of
bacterial and development of natural microbicides containing these peptides as active ingredients. N- to C-terminal
cyclic peptides have an inherent stability towards degradation by proteases, although they remain biodegradable,
making them exceptional candidates for eco-friendly microbicides and longer term protection of plants, plant material
and products against the slow growing fungal pathogens. We focused in this project on the TRCs, as they have thus far
shown promising antimicrobial activity toward numerous pathogenic microorganisms which cause food spoilage and
disease including: the Gram positive bacteria Listeria monocytogenes (Spathelf & Rautenbach 2009), as well as a
multitude of pre- and post-harvest fungi such as Botrytis cinerea, Fusarium spp and Penicillium spp (Troskie et al. 2014a),
as well as the human malaria parasite Plasmodium falciparum (Rautenbach et al. 2007). All of which directly or
indirectly result in extensive morbidity and mortality in developing countries such as those of Southern Africa.
3. AIMS AND OBJECTIVES / RESEARCH QUESTIONS
Pre-harvest and post-harvest production losses in the agricultural sector, as a consequence of microbial disease, are
serious problems which severely impacts productivity and global food security. With the existing antimicrobial agents
losing potency towards many of the pathogens due to emerging resistance, the increased global opposition to the use
of chemical control and the movement towards more natural/organic farming practice, there is an immense need for
natural bio-control agents. A large group of natural bio-control/microbicide agents already exists, namely the
antimicrobial peptides that are part of the first line of defence of all living organisms. The research question is: Can
antimicrobial peptides serve as safe green microbicides to protect plants from the seed, graft, cutting or tissue culture
phase through to the established young plants in nurseries and final products in the market?
The primary goal of this translational research project is to obtain “proof of concept” for the application of natural
peptide microbicides with the tyrocidines as model group to (1) prevent latent fungal pathogen carry-over into plant
culture/nursery propagated plants and (2) afford protection against post-harvest fungal pathogens infecting harvested
produce. The long term goal of the study is to develop and economically produce environmentally friendly,
biodegradable microbicide(s) based on naturally produced antimicrobial peptides for the agricultural and food
industry.
Phase I
Research aim – Optimisation of natural peptide production/purification and design of tailored natural peptide microbicide formulations
Objectives 1-4
1. Design and optimising fermentation of producer cultures to reach small field trial production levels of selected
antimicrobial peptides and complexes.
2. Up-scaling and optimising economical purification of selected antimicrobial peptides and complexes for
formulation and field trials.
3. Tailoring natural peptide microbicide combinations for sterilising packaging materials, neutralising the causal
agents of plant culture/seed/cutting infections, woody plant graft failures, post–harvest infections and premature
cut flower decline.
4. Development and testing of formulations of the selected antifungal peptides and combinations to produce
natural peptide microbicide formulation(s) for applied in vitro and in vivo testing.
Milestones 1-3
1. Economical optimised production and purification of 0.1-0.5 kg natural antimicrobial peptide(s), including an
techno-economical prediction model (Objectives 1 and 2).
2. One or more tailored peptide microbicide combinations for sterilising packaging materials, neutralising the causal
agents of infection in plant cultures, woody plant graft failure, post–harvest infections and premature cut flower
decline (Objective 3).
Natural Antimicrobial peptides in Agriculture
3. Natural peptide microbicide formulations for different agricultural applications with long term stability and activity
(Objective 4).
Phase II
Research aim – Proof of concept in vitro and in vivo testing of natural peptide microbicide formulations in agricultural/industrial applications
Objectives 5-10
5. In vivo evaluation of peptide microbicide formulation(s) toxicity on bees and nematodes.
6. In vivo evaluation of peptide microbicide formulation(s) to prolong the vase life of selected flowers from microbial
infections.
7. In vitro assessment of the surface activity of peptide microbicide formulations(s) on different materials such as
wood, paper and plastic polymers.
8. In vivo evaluation of peptide microbicide formulation(s) to protect selected seasonal fruits from post-harvest
fungal infection against plant pathogens in simulated storage environments.
9. In vivo evaluation of peptide microbicide formulation(s) in plant culturing and micro-propagated plants.
10. In vivo evaluation of peptide microbicide formulation(s) to protect woody plant cuttings and grafts against
fungal plant pathogens in nursery environments.
Milestones 4-9
4. Natural peptide microbicide formulations have no toxicity towards honey bee at the highest concentration used
in field trials (Objective 5).
5. Natural peptide microbicide treatments significantly increasing the vase life of selected cut flowers by lowering
the decline due to infection versus controls (Objective 6).
6. Protection of selected materials against infections/biofilms by selected microbes (Objective 7).
7. Natural peptide microbicide washing treatments significantly lowering the infection rates/rotting of fruits versus
controls (Objective 8).
8. Natural peptide microbicide treatments lead to significantly higher yields and survival of healthy viable explants
from plant cultures and micro-propagated plants than controls (Objective 9).
9. Natural peptide microbicide treatments significantly benefited the young nursery propagated woody plants with
higher or similar survival versus controls treated with conventional chemicals. (Objective 10).
4. LITERATURE REVIEW
Plant diseases caused by fungal infections are a major contributor to quality failure and loss of agricultural products
(Montesinos 2007). The effective and durable pathogen control in agriculture is one of primary goals in modern
agriculture (Kaur, Sagaram & Shah 2011).
A major concern in the agricultural industry is pre-harvest losses that are mostly caused by decline of plant health due to
infection. Soil-borne Fusarium spp are associated with vine wilt and root rot and affect many plant species (Di Peitro et
al. 2003; Berrocal-Lobo & Molina 2008; Highet & Nair 1995). Fungal spores can persist in soil for years and cuttings for
planting or leaf detachment can encourage infection through vascular wounds, although infection in many plants
takes place through roots (Berrocal-Lobo & Molina, 2008; Highet & Nair, 1995). However, the two most destructive
diseases associated with grapevine decline are black foot disease, caused by Cylindrocarpon spp, and young
grapevine decline or Petri disease caused by Phaeomoniella spp. and Phaeoacremonium spp (Fourie & Halleen 2002,
2004). Black foot disease and Petri disease primarily infect grapevine propagation material and newly planted vines
and are either individually or collectively responsible for the decline of young vines, reduction or loss in productivity and
young vine death. Older vines that have been infected with these diseases show a stunting phenotype and low or even
no fruit carrying potential. As a result, grape farmers are forced to replant young infected vineyards at a substantial cost
and loss of production. The problem, however, often arises at the nurseries that supply the propagation material.
Research has shown that the primary source of infected material is mother block material and nurseries, with less than
50% of propagation material yielding healthy saleable plants (Fourie and Halleen 2004; Halleen, Crous & Petrini 2003).
Halleen, Fourie & Crous (2007) evaluated 13 fungicides, representing 10 different chemical classes for in vitro mycelial
inhibition of Cylindrocarpon spp and Campylocarpon spp, the causal agents of black foot disease. Only prochloraz
manganese chloride, imazalil and benomyl were able to effectively reduce mycelial growth in all fungal strains tested,
Natural Antimicrobial peptides in Agriculture
but failed to protect propagation against black foot disease under field conditions. This study showed that most of the
chemical treatments were ineffective and inconsistent in protecting grapevine against black foot disease. Many other
fruit producing woody plants are propagated via grafting and are also plagued with plant specific infections leading to
graft and orchard losses. Peaches can be plagued by fungal infections targeting the whole plant such as Brown rot
(Monilinia fructicola) or the trunck such fungal gummosis (Botryosphaeria dothidea), whereas examples of truck diseases
in appels are Bot rot (Botryosphaeria ribis) and Collar rot (Phytophthora cactorum) (Texas Extension Plant Pathologists
n.d.). Benomyl was regularly used as fungicide for these diseases, but due to wide spread resistance benomyl and
analogous compounds are largely ineffective (Ma, Yoshimura & Michailides 2003; Leroux & Clerjeau 1985)
Plant tissue culture is an important technique in the grapevine, fruit, flower and plant industry for the supply of virus- and
pathogen-free planting material for the establishment of new vineyards and orchards. A second tissue culture
technique, namely embryo rescue, plays an important role in the breeding of new cultivars, especially seedless varieties.
Although these techniques have been well established in supplying good quality planting material there are still a
number of aspects that influence the success rate of establishing field material, into an in vitro environment. One of the
most important factors is explant quality, which can be greatly affected by the microbial population (fungi, bacteria
and yeasts) present on field plant material (Leifert & Cassells 2001). The rich medium used to establish explant material in
vitro is also a perfect medium for the growth of these microbes (Leifert & Cassells 2001), making the sterilization strategy
which is employed a crucial step in the successful establishment of explant material from the field back into in vitro
culture. Sterilization of explant material usually involves a combination of hot water treatment, alcohol and NaOCl2
treatments (Cassells 2000), but this only removes some of the surface microbes, while endophytic bacteria and fungi
survive, leading to contamination when the explant is placed on the culture medium. Tissue culture laboratories rely on
antibiotics to control both fungal and bacterial infections, but it is rarely the case that a single microbe is present on the
explant material, requiring combinations of antibiotics, which can lead to phytotoxicity (Estopá et al. 2001). PPMTM,
containing methylchloroisothiazolinone/ methylisothiazolinone as active ingredients, is currently the only chemical
product on the market that claims to inhibit the growth of both bacterial and fungal pathogens, but it has been
reported to interfere with plant development, especially in grapevine tissue culture (Compton & Koch, 2001).
In agriculture, post-harvest losses are in the region of 24% and about 50% in underdeveloped tropical countries
(Courtsey & Booth 1972). Post-harvest infections, such as those caused by Penicillium spp (blue mould), Monilinia spp
(brown rot) and particularly Botrytis cinerea (grey mould) in grape, strawberries, cherries and tree fruits like pears and
apples, are the leading cause of major losses in marketable fruits (Lennox 2003, Wilson et al. 1991 Romanazzi 2010).
Measures to limit losses during storage include low temperature storage, bio-control and treatments with natural
compounds as well as chemical treatments such as chemical sprays, dips or washes and fumigation (Zheng, Yang &
Chen 2008, Romanazzi 2010) and specialised packing (treated wrappers, box liners or shredded paper).
Chemical treatments include the use of borax, sodium ortho-phenylphenate, chlorine, antibiotics, diphenylamine and
ethoxyquin. Fumigation includes the use of sulfur dioxide, nitrogen trichloride, ammonia or ammonium compounds and
carbon dioxide. Packaging material is treated with biphenyl, orthophenylphenol, iodine, copper sulphate, mineral oil
and diphenylamine. Orthophenylphenol impregnated wrappers have been shown to be effective against some citrus
fungi and lower the infection of tomatoes, grapes and apples (Van der Plank, Rattrey & Van Wyk 1940). However, injury
and scalding of the fruit was observed after exposure to orthophenylphenol impregnated wrappers. Iodine
impregnated wrappers have also been shown to have activity against blue mould without damage to citrus, but with
the drawback that iodine’s volatile nature causes the inhibitory effect to wear off quickly (Smith 1962). There are still
other treatments preventing fungal infections, but with a range of drawbacks, of which damage to the fruit and
vegetables is most prominent. Most of these chemicals are unnatural and may lead to unwanted chemical residues
and have also environmental issues with its production and waste management. Also with the more stringent EU
legislation on pesticides, biocides and chemical residues on produce (Williams 2011) producers are faced with the
problem of limited agents that can be utilised. As a result cold storage is generally required to limit infections, but some
pathogens can still grow on the fresh produce at low temperatures, as most of us have experienced in with fruit and
vegetable spoilage in our fridges at home. There is therefore not only a need for a safe microbicide, but because there
are always microbial pathogens in the environment there is still a need for a potent natural antimicrobial impregnated
packaging that will not lead to damage of fruits and vegetables or leave an unwanted chemical residue. The persistent
post-harvest losses and resistance to treatments used to protect fruit during storage (Spotts & Cervantes 1986) are
serious problems that must be addressed in the light of consumer resistance to foreign chemicals in food and
beverages. Similar issues concern other fresh produce such as cut flowers, where microbial infections during the post-
harvest period lead to large product losses.
One of the biggest problems in the food industry is bio-fouling and once an organism has adhered and colonised to a
surface, it can form resistant biofilms that are difficult to remove completely, leaving a constant source for re-infection or
chronic bio-fouling. In the food industry, Listeria monocytogenes is commonly associated with bio-fouling and found in
meat and dairy processing plants (Poimenidou et al. 2009). Although the sheer force used to clean pipes within the
processing plants should be enough to remove exposed biofilms, it is the hard-to-reach places (such as cracks within
Natural Antimicrobial peptides in Agriculture
equipment caused by age, gaskets, valves and joints) that are more likely to develop biofilms and these are difficult to
remove. Furthermore, environmental surfaces (floors, walls etc.) are found to be subject to extensive biofilm formation
and can lead to reintroduction of Listeria into a cleaned processing plant. In conjunction, resistance of Listeria spp to
sanitizing agents used within the food processing environment has been observed. This is of great concern, since Listeria
spp is responsible for 28% of deaths caused by the intake of contaminated food in the USA (Mead et al. 1999).
The conventional antimicrobial agents used in agriculture and food preservation are losing potency towards many of
the pathogens due to resistance (McManus et al. 2002; White et al. 2002). In agricultural research there has been lately a positive drive towards using microorganisms as bio-control agents, although it was already studied in the mid-1900s
Weller et al. 2002). At the height of chemical control of plant disease in the 1980’s it was shown that Bacillus subtilis B-3,
an iturin A peptide producer, control brown fruit rot (Monilinia fructicola) of stone fruits and also a spectrum of other
phytopathogenic fungi (Pusey & Wison, 1984; Pusey, 1989). Bacillus subtilis RB14, a producer of the both the peptides
iturin A and surfactin, inhibited the damping-off of tomato seedlings caused by Rhizoctonia solani (Asaka & Shoda
1996). The surfactin producer, Bacillus subtilis 6051 have been shown to have important rhizosphere functions, by forming protective biofilms and inducing systemic resistance in plants (Bais, Fall & Vivanco 2004).
In microbial bio-control there is an inherent unpredictability in terms of the growth of the microorganism and production
of the antimicrobial peptide (AMP) under agricultural conditions. An ever changing agricultural environment and poorly
controlled application of a variety of commercial microorganisms, of which some have antagonistic action towards
each other, have led to the impression that the efficacy of microbial bio-control is unpredictable and there is no
guarantee of protection against all the causative pathogens. Therefore, in certain applications it may be more secure
to use the isolated AMP to control pathogens. Alternative strategies include expressing the AMPs in the genetic
manipulated (GM) plants to increase their resistance towards pathogens, but the consumer resistance to GM products
has limited general application of this technology. However, many cyclic AMPs from naturally occurring soil organisms
offer the possibility of economical production by microbial cultures, as well as longer term stability due to their stabilised
cyclic nature.
In nature AMPs is a universal group of natural bio-control agents that have been underexploited as natural bio-control
agents. Many of the AMPs have a broad spectrum of activity against bacteria (Gram-, Gram+ and/or mycobacteria),
fungi, parasites and certain viruses (Jenssen et al. 2006; Rautenbach & Hastings 1999). A large degree of structural
diversity is found in the collection of AMPs, but they share one important feature–their amphipathic character.
Consequently, many of these AMPs act upon cell- and other membranes, causing disruption of membrane function, as
well as rapid death of the target cell. Their basis for selectivity appears to be related to the composition of the target
membrane (Melo, Ferre & Castanho 2009). There is mounting evidence that many AMPs also have sensitive cell wall
and cytosolic targets (protein/nucleic acid/metabolite) (Zhang et al. 1999; De Lucca & Welsch 1999). Their rapid killing
kinetics, multiple targets and lack of natural resistance most probably led to the evolutionary selection of this group as
universal defence molecules.
In medicine AMPs with antifungal activity have been considered as a potential source of a new class of fungicides (De
Lucca & Welsch 1999; Ajesh & Sreejith 2009). The recorded antifungal peptides share the structural diversity with the
broad group of AMPs, but two structural groups are particularly well represented namely cyclic peptides and cyclic
lipopeptides (De Lucca & Welsch, 1999). These peptides are inherently more resistant to proteases due to their cyclic
nature and are generally produced by microorganisms, of which many are natural soil organisms that could be used as
agricultural bio-control agents. As mentioned above, bacterial producers of the iturins, a group of cyclic lipopeptides,
was successfully used in bio-control. Iturin A has been shown to act synergistically with the cyclic lipopeptide surfactin,
which is co-produced by some Bacillus subtilis strains (Maget-Dana et al. 1992; Thimon et al. 1992). Apart from the iturins,
many of the cyclic lipopeptides and cyclic peptides such as the aculeacins, aureobasidin A, cepacidines,
echinocandins, neumocandin, schizotrin, syringomycin, syringostatin, syringotoxin were shown to have potent activities
towards Candida spp or Aspergillus spp with diverse targets such as beta-glucan synthesis, actin assembly and
membranes (lysis) (De Lucca & Welsch 1999). It is therefore quite possible that these peptides may have activity against
fungal plant pathogens. Another cyclic peptide that has potential as bio-control agent is the cyclic decapeptide GS
(Murray, Leighton & Seddon 1986). The GS producer was shown to have protective activity against Fusarium oxysporum on tomatoes (Chandel, Allan & Woodward 2010) and both GS and it producer exhibited protective activity against
powdery mildew in cucumbers (Schmitt et al. 1999). A broad spectrum of antibacterial activity has also been reported
for GS (Kondejewski et al. 1996). However, previous studies on the TRCs, with 50% analogy to the cyclic peptide GS,
were limited to two reports concerning their antifungal activity (Mach & Slayman 1966; Trevillyan & Pal, 1979) on the
potent activity on Neurospora crassa and one report on the activity of tyrocidine-gramicidin complex, tyrothricin (TCN),
on Candida albicans (Kretschmar et al. 1996). Our group were the first to show the broad spectrum activity of the TRCs
towards phytopathogens, in particular filamentous plant fungi (Troskie et al. 2014a). The TRCs are cyclic decapeptides,
analogous to GS, containing one of the pentapeptide repeats of GS. TCN was the first antibiotic to be used in clinical
practices (topical applications), but was soon replaced by penicillin (Dubos and Cattaneo 1939; Hotchkiss and Dubos
Natural Antimicrobial peptides in Agriculture
1941, Bradshaw 2003). The TRCs, as well as their structural analogues the tryptocidines and phenycidines (Fig. 1), are
isolated from the TCN peptide complex which is primarily produced during the late logarithmic growth phase by the soil
bacterium Brevibacillus parabrevis (also known as Bacillus aneurinolyticus and previously classified as Bacillus brevis)
(Dubos, 1939, Marahiel, Nakano, & Zuber 1993).
The TCN complex is composed of two fractions, the first being a neutral fraction consisting of linear pentapeptides
known as the gramicidins (GRMs) and the second being a basic fraction consisting of cyclic decapeptides, namely the
tyrocidines or TRCs (Tang et al. 1992).
D-Phe1
Pro2
Phe3
(Trp3)
D-Phe4
(D-Trp4)
Leu10
N HN
O O
O
NH
O
NH HN
O
HN
O
Orn9
(Lys9)
H2N
O O HN
NH HN
NH
O
O H2N
O
O
Asn5
Val8 H2N Gln6
Tyr7
(Trp7
OH Phe7)
Figure 1: The chemical structure of tyrocidine A, one of the major TRCs. Conventional three-letter abbreviations are
used for amino acid residues, except Orn for ornithine. The alternative amino acid residues for the other
peptides in the TRC complex are indicated at positions 2, 6, 7 and 10. Lys in position 2 leads to A1, B1 and C1
analogues. Phe in positions 3 and 4 leads to the A, A1 analogues, Trp in position 3 to the B, B1 analogues and
Trp in 3 and 4 position to the C, C1 analogues. Tyr in position 7 leads to the tyrocidines, Trp to the
tryptocidines and Phe to the phenycidines (adapted from Tang et al., 1992).
TCN, containing the TRCs and GRMs, was the first antibiotic preparation to be used in clinical practices, despite being
discovered 10 years after penicillin (Dubos 1939), yet due to its haemolytic toxicity its use was limited to topical
applications (Bradshaw 2003) such as Tytin® and in throat lozenges such as Tyrozets®. Consequently attention has been
focused on the β-lactam antibiotics as chemotherapeutic agents.
Our group’s recent research brought the true agricultural potential of the natural tyrocidines as green microbicides and
plant stimulants to the fore. Our group showed that the purified TRC peptides and natural TRC peptide complexes have
highly potent activity on both spore and hyphae of a broad range of agronomically important fungal phytopathogens
(Troskie et al. 2014a), as well as antibacterial activity against a range of bacterial pathogens (Leussa and Rautenbach
2014). The applied research of green microbicide development was only initiated during the last three years and
expanded with the support the Green Fund Grant RW1/1160. Vosloo et al. (2012) proved that it is possible to produce
the TCN peptides and manipulate the TRN and TRC profiles in bacteria cultures, as well as produce more than one gram
of peptide per litre of culture. Economical fast small scale isolation protocols and high performance liquid
chromatography (HPLC) protocols for the analysis/purification of peptides in the antimicrobial peptide complexes was
specifically developed for the TRCs and GRMs (Eyéghé-Bickong 2011). With this basis we endeavoured on this project as
“proof of concept” study for the application of natural peptide microbicides with the tyrocidines as model group.
gure 2 The strategy outline of the approach followed in this project on “Natural antimicrobial peptides as green
Natural Antimicrobial peptides in Agriculture
5. METHODOLOGY In our research we follow a general methodological approach of peptide discovery, characterisation and subsequent
production of most promising peptide product candidates, followed by application in in vitro and in vivo trials. The
scheme in Fig. 2 gives an overview of our approach in this project with the green spheres the focus of the research
reported here.
Fi
microbicides in agriculture: A proof of concept study on the tyrocidines from soil bacteria”.
Phase I
Up-scaling and optimisation of natural peptide production/purification and design of tailored natural peptide microbicide formulations
Research Purpose and Design
In order to economically produce the active ingredients in the proposed green microbicides, namely natural
antimicrobial peptides, it is necessary to design the optimal fermentation and isolation procedures. For this initial study
we focused of the production of the TCN complex by the soil bacterium Brevibacillus parabrevis. We aimed to design
the whole process (production and isolation) to limit the energy and waste footprint when the process is used on an
industrial scale.
Natural Antimicrobial peptides in Agriculture
Design and optimising fermentation of producer cultures
Design of the fermentation depends on the prerequisites of the culturing of the bacterial producer. Optimised small
volume production (20 mL to 200 mL) of the selected antimicrobial natural peptide complexes and combinations in
flask cultures was adapted 10 x 2 L fermentations in a customised fermentation set-up. The selection of bacterial
producer colonies was optimised by culturing producer bacteria/yeast on agar plates containing indicator organism
and on blood agar plates. The medium base (salts, sugars), N-source(s), metal ions, pH, oxygenation and culture time
was adapted to optimise production and focus production from complex mixtures to tailored peptide complexes.
Peptides were harvested from biomass samples using established extraction procedures and analysed electrospray
mass spectrometry (ESMS) and ESMS linked to ultra-performance liquid chromatography (UPLC-MS). Culture
composition and conditions, peptide yields and identities, as well as antifungal and antibacterial activity of the peptide
extract were documented as part of optimisation quality control.
Up-scaling and optimising purification for formulation and field trials
Optimisation of the small scale IP protected extraction protocols for fast and economical preparation of semi-pure
antimicrobial peptide fractions (>40%, >75% and >85% purity). The produced peptide was extracted according to an
optimised method based on the original extraction methods (Dubos & Hotchkiss 1941, 1942; Hotchkiss & Dubos 1941).
The optimised purification method of crude peptide can only be briefly described since it is currently protected under a
non-disclosure agreement (NDA) as it has been classified as trade-secret (BIOPEPTM, University of Stellenbosch). The
biomass was extracted using an extreme pH step, organic solvent extractions, precipitation steps and/or activated
carbon treatments, followed by chromatographic purification. This yielded crude extracts of about 50% and purified
peptide fractions with >75 and >85%. The purified fractions were chemically characterised using analytical RP-HPLC and
UPLC-MS (Vosloo et al. 2012). From these laboratory and medium scale production/isolation data, the costing was
calculated and utilised as the basis for preliminary financial predictors for commercialisation.
Tailoring natural peptide microbicide combinations
Activity determination of purified peptides and in combinations and formulations was done using antifungal and
antimicrobial assays described by Troskie & Rautenbach (2012) and Du Toit & Rautenbach (2000), respectively. From
these activity parameters the most potent antimicrobial combination of peptides in the formulations were chosen for
the different formulations. Each formulation was prepared considering the TRC content, additive and specific
application.
Development and testing of selected antifungal peptide formulations
Amphipathic peptides tend to aggregate over time in aqueous environments which leads to activity losses. Therefore
the optimal peptide formulation was determined for use in applied in vitro and in vivo studies must be obtained. The
formulations were designed to keep the peptide active and in solution, assist in longer retention of surfaces and/or
boost natural plant defence. The influence of peptide purity, peptide stock solution concentration and different solvent
components (ie organic/aqueous solvents and mineral salts) was determined in terms of the biophysical character
(aggregation status) of the peptide component in solution using dynamic light scattering and activity in diluted form.
The activity of most active peptide preparations in the presence of natural saccharide-type formulations was
determined against target bacteria and fungi by in vitro testing, using conventional and adapted activity assays
(Troskie & Rautenbach, 2012; Du Toit & Rautenbach, 2000).
Phase II
Proof of concept in vitro and in vivo testing of natural peptide microbicide formulations in agricultural/industrial applications
Research Purpose and Design
Proof of concept assessment of tailored natural peptide microbicide formulations(s) for sterilisation of plant/food
packaging materials, plant material and culture media used in plant cell cultures, seedling, cuttings, protect woody
plant grafts from fungal infection in nursery environments, to protect selected seasonal fruits from post-harvest fungal
infection and to prolong the vase life of selected cut flowers by preventing microbial infection.
In vivo evaluation of peptide microbicide formulation(s) toxicity on insects
In vitro determination of the natural peptide microbicide formulation toxicity was done using human erythrocytes
(spectrophotometric haemolytic assay) and cytotoxic activity assays using Spodoptera frugiperda (Sf9) insect cells and
resorufin-resazurin (also known CellTiter Blue or Trypan Blue) assay (Rautenbach et al. 2007). Determination of toxicity
towards against Caenorhabditis elegance, bee larvae (Apis mellifera carnica) and bees (Apis mellifera scutellata) was
Natural Antimicrobial peptides in Agriculture
to assess the relative safety of the natural peptide formulations at the highest concentrations use in field trials. The
toxicity of TrcA was evaluated using the Caenorhabditis elegans model system described by Breger et al. (2007) in
collaboration with Katholieke Universiteit Leuven (Leuven, Belgium). The acute oral toxicity of the TCN75 formulation,
which was to be utilized within agricultural applications, was evaluated toward adult honey bees using methodology
described in OECD/OCDE Test Guideline 213 with dimethoate (DiM) as positive toxicity control. The in vivo toxicity of
tyrothricin formulations toward Apis mellifera larvae was tested according to the OECD/OCDE Test Guideline 237 in
collaboration with the Technische Universität Braunschweig (Braunschweig, Germany).
In vivo evaluation of peptide formulation on the selected vase flowers
Determination of the extension of vase life by the peptide formulations was done on freshly cut flowers (Gerbera and
Delphinium) under simulated vase conditions. In order to obtain reliable results and eliminate any variability in terms of
flower quality, flower maturity and exposure after cutting, the flowers used for the trials needed to be obtained directly
from the suppliers/nurseries. Freshly cut African daisies (Gerbera hybrids) flowers and blue larkspurs (Delphinium hybrid)
were obtained directly from the nursery and immediately placed in water containing the different treatments. Since
different species may react differently to treatments, three different Gerbera species were used in the trials, Gerbera
larreia, Gerbera mermaid and Gerbera florade. Freshly harvested cut flowers was put into either tap water, tap water
containing peptide formulation (25 and 50 mg/L), commercial product (Comm A or Comm B) or a mixture of the
peptide formulation and commercial product. The decline of the flowers was visually monitored and documented over
8-18 days. Flower condition was rated from 0 to 5, with 0 = dead, 1 = flower petals dehydrated, closed and drooping, 2
= flower still partially open but dehydrated and drooping, 3 = flower starts drooping, petals soft, 4 = some petals
becoming soft, flower still in good condition and 5 = flower is in pristine condition with taught petals and stem/leafs.
In vitro assessment of the surface activity of peptide microbicide formulations
Materials such as wood cuttings, paper and paper products and a selection of synthetic polymers and nanofibres were
treated with >75% purity TCN peptide formulations. The TCN treated materials were then challenged with a selected
fungal strain or bacterial strains (Listeria monocytogenes and Micrococcus luteus) to assess the antimicrobial properties
of the material. The growth of the microorganism on/around the treated and untreated materials was visually
documented or determined via an adapted Trypan Blue assay. Cellulose was selected as material for further analysis on
the robustness of the antimicrobial activity and subjected to washes with water at different temperatures, 2% NaCl, 50
and 100% acetonitrile and solvents with pH ranging from 1-13. Treated cellulose was also assessed for maintenance of
activity over time.
In vivo evaluation of peptide microbicide formulation on seasonal fruits
Determination of the protection by the peptide microbicide(s) of selected seasonal fruits (citrus and strawberries) was
done with Penicillium spp or Botrytis cinerea as fungal pathogens (depending on fruit) under storage conditions.
Whole fruits (strawberries) were painted with tap water lased with fungal spores after dip treatment with either natural
peptide formulation or with water treatment (control). Alternatively, wounded fruits (oranges, trail in collaboration with
ICA) was challenged with fungal spores and then washed in peptide treated or control biocide (Comm C) treated tap
water. The infection and spoilage of fruits, stored in covered trays at 40C and ambient temperature, was monitored over
7 days. Evaluation of protection was done by visual detection of infection/spoilage.
In vivo evaluation of peptide formulations in selected plant cultures and micro-propagated plants
Sterilisation of tissues for explants from field contaminated Vitis vinifera or grapevine (model woody plant) specimens
was done using the >85% TCN peptide microbicide formulation(s) and/or standard water/ethanol/NaOCl2 for washing
steps. The >85 TCN formulation(s) was included in, or omitted from the plant culturing media. The plant cultures were
evaluated after one and two months on the grounds of residual microbial contamination, plant culture survival and
growth. For the plant toxicity studies Arabidopsis thaliana was chosen as model non-woody plant. Seeds were allowed
to stratify on rehydrated Jiffy-7®-peat pellets for three days in the dark. The seeds were then transferred to growth
chambers where they germinated and developed. The peat pellets were either treated with water or >75% TCN
formulation (1 x 50 g or 2 x 25 g). Visual examination of plants was done after 5 weeks and germination, roots and
leafs were evaluated and documented.
To study the effect TCN has on the germination of tomato seeds (Money Maker, Starke Ayres) the normal damp
cellulose filters used in germination were replaced by cellulose filters that were treated with a 5 mg/L, 25 mg/L or 50
mg/L solution of a >75% TCN formulation. The tomato seeds were washed in 70% ethanol followed by a
decontamination step in 0.35% NaOCl2 (bleach) and finally a water wash. Each of the prepared petri dishes with either
treated or untreated cellulose contained 25 seeds per dish and was monitored over an 8 day period for microbial
contamination and germination. Finally the young geminated plants were analysed for biomass, as well as shoot and
root length.
Natural Antimicrobial peptides in Agriculture
In vivo evaluation of peptide microbicide formulation on woody plants
Grapevine grafting was done in a normal nursery setting (Fleury nursery, Wellington and Stargrow nursery, Citrusdal),
where grafts were treated with 50 mg/L of selected TCN peptide formulation. The grapevine grafts were dipped and
treated using the conventional methodology used by the nursery and planted in field. The young grapevines treated by
the natural peptide formulation and by commercial biocide (Comm C) were evaluated for germination after three
months by manual counting. The harvesting and grading of the young grape plant material (“sticklings”) was done
using conventional methodology by the respective nurseries.
A late season and in season peach grafts trial in collaboration with Rosenhof nursery (Ceres) was conducted to assess if
the TCN formulation could overcome late and in season losses. The normal nursery practice was followed, but for
adding a dipping step with the TCN formulations.
In season blackberry cuttings trials in collaboration with Rosenhof nursery (Ceres) was conducted to assess if the TCN
formulation could overcome losses. The normal nursery practice was followed, but adding either a dipping step with the
TCN formulations or a direct addition step of the TCN formulation to the planted cuttings.
An in season apple cutting trial in collaboration with Stargrow nurseries at Suikerbosrand, Koue Bokkeveld and Citrusdal
was conducted with a cultivar that routinely gives very low yields to investigate if the TCN formulation could improve the
low yields normally observed. The normal nursery practice was followed with the addition of a TCN dipping step.
6. CHALLENGES AND CONSTRAINTS
We experienced six main challenges that placed constraints on the rate of project progression, namely
1. Human resource procurement and development were particularly difficult, as research personnel with training in
the scarce skills needed in this project are not willing to commit only to a 12-18 month contact. This issue led to the
loss of Dr Anscha Troskie, one of the principle researchers on the plant culture trial and fruit/cut-flower protection
section of the project, after 12 months. She was recruited into the job market as scientist at Kapa Biotech. The 18
months period starting in April is also too short for a post-graduate project and we could only enrol current students
with projects that fell in the scope of this project, as well as short term interns.
2. Set-up of the advanced analyses infrastructure and medium scale production facility was delayed by more than 6
months due to slow import and shipping, as well as SA customs and SA Reserve Bank delays during the
procurement.
3. Laboratory space for the upscaling is too small to accommodate the apparatus for medium scale culturing,
production and purification of the antimicrobial peptides for our trials. Extra laboratory space was negotiated with
the Department of Biochemistry and the large instruments were accommodated in secure laboratories with all
occupational safety measures in place, including emergency power. However, this is a short term arrangement
and not ideal as these instruments need to be placed in a specific configuration to ensure optimal production and
purification. Three of these instruments are only dedicated to upscaling, which is not trivial and unfortunately yields
low scientific outputs. Such endeavours are not conducive to postgraduate training and scientific output
dependent funding.
4. Agricultural and nursery field trials on unknown natural products are difficult to organise, especially with the majority
of current agricultural sector practices about financial gain and not always concerned with environmental issues.
One small trial that deliver unconvincing results could lead the nursery to discard such a treatment and not be
open to conduct follow-up trials.
5. Field trials in agriculture are labour, infrastructure and material intensive and each one takes 2-12 months to deliver
results, depending on type of trial, as it is dependent on plant growth.
6. Field trials in agriculture are risky, as they are depended on multiple parameters that the researcher cannot control,
such as agricultural practice, biological and ecological variability and weather. Due to the multiple parameters,
trials must be run for 3-5 seasons to ensure data that could be statistically evaluated. During the 20 months we were
only able to complete three separate trials and we will have to continue this research for at least three more years
to get publishable results.
Natural Antimicrobial peptides in Agriculture
7. RESULTS/FINDINGS
Phase I Research Results
Design and optimising fermentation of producer cultures
The research on the design and optimising of fermentation of producer cultures formed part of the PhD thesis of JA
Vosloo, Department of Biochemistry, Stellenbosch University. Due to future commercial applications of this part of the
project very little detail of the production and purification methodologies can be revealed. The parameters and best
media for the optimised productions in fermentation vessels have been determined and are protected under NDAs with
all members working on these fermentations order to insure the future commercialisation potential of the peptide
products. However, the overall production strategy without the full experimental details is given in Fig. 3 and discussed
below.
Variable tyrothricin (TCN) production levels not only among different strains of the TCN producers, but also between
colonies of the same strain have been observed (Dubos 1939; Dubos & Hotchkiss, 1941, 1942; Lewis, Dimick & Feustel
1945). In the present study and in literature (Dubos & Hotchkiss, 1941), the change in total TCN production and colony
morphology was observed with successive culturing on agar media. The maintenance of high producing colonies was
found to be instrumental for maximal tyrothricin production to occur. Increased tyrothricin production within stationary
cultures is proposed to be dependent on the utilization of complex nitrogen sources (Appleby et al. 1947). Variations in
media composition as well as the culturing conditions were performed in an effort to elucidate the optimal environment
in which Brevibacillus parabrevis maximally produced tyrothricin. Four different media compositions were used and the
tyrothricin yield and peptide profile was assessed (Table 2).
Table 2: Summary of the average tyrothricin yield from cultures of Brevibacillus parabrevis grown in different media.
Culture Medium
(Fermentation Time) Media character
Crude extract
mass ± SEM
(g/L)
% Tyrocidine
in extract*
Calculated amount
of tyrothricin (g/L)**
Medium A
(10 days)
Medium B
(10 days)
Medium C
(10 days)
Medium C
(17 days)
Medium D
(10 days)
Mixed animal and
digested protein + urea
High digested animal
protein content
Glucose + digested
milk protein
Glucose + digested
milk protein
Glucose + digested
milk and plant protein
0.32±0.03
(n=20) 19 0.08
0.35±0.05
(n=7) 15 0.07
2.80±0.17
(n=45) 40 1.49
3.32±0.50
(n=4) 40 1.77
2.40±0.20
(n=7) 40 1.28
SEM, standard error of the mean; * determined via UPLC-MS, calculated relative to commercial tyrocidine mixture; ** Calculated
from the expected 20:20:60 ratio of contaminants:GRMs:TRCs in the crude tyrothricin (Hotchkiss & Dubos, 1941, Dubos & Hotchkiss,
1942).
The natural production of TCN, containing the linear GRMs and cyclic TRCs, by Brevibacillus parabrevis was increased to
nearly 2 g/L medium through the elucidation of the optimal production medium (Medium C), as well as growth
conditions. This high production made it highly feasible to upscale the peptide production in order to reach a
production scale necessary for agricultural trials.
Up-scaling and optimising economical purification
This research on up-scaling and optimising economical purification formed part of the PhD thesis of JA Vosloo,
Department of Biochemistry, Stellenbosch University. We have exceeded our lower limit 0.1 kg of production and nearly
0.4 kg TCN was extracted because we were able to consistently obtain high yields of TCN via our optimised culturing
strategy. Moreover, culture batches were up-scaled from 200 mL (maximum 2 L) to 2 L batches with maximum of 10 L
total volume per fermentation or 30 L/month, with an expected yield of 50-60 g of peptide/month. A further increase in
culture volume is possible, however, we have reached the required amounts to run several large in vivo field trials with
the crude extracts containing 40% TRCs or >65% TCN peptides (Fig. 3, Table 2). These extracts were found to have at
least 70% activity at the same mass-based concentration as a highly purified commercial TRC mixture. For the
application of peptides in plant cultures, micro-propagation of plants, sterilisation of fruits and to create antimicrobial
materials, we developed and optimised two routes of purification of the tyrocidines (Fig. 3).
Natural Antimicrobial peptides in Agriculture
Figure 3: Flow diagram of the production and purification steps used in the optimised production of TCN and TRC
preparation.
First, the tyrocidines are extracted out of the cells using organic extraction. They are subsequently taken through a
number of steps where their solubility is manipulated to remove contaminants (purification step 1 a/b/c) to yield >75%
pure extracts (Fig. 3). These extracts were found to have > 85% activity at the same mass based concentration as a
highly purified commercial Trc mixture against bacteria. This activity is higher than expected and is probably due to the
natural pigment contaminants which are acting as chaotropic agents. Such agents can limit the aggregation that
leads to loss of activity in the highly purified samples.
As the initial steps involved of culture extraction, as well as purification step 1 (a, b and/or c) involving up to 10 L media
or solvent volume per extraction and purification step, this entailed a 50 times upscaling from our laboratory scale of
handling 200 mL at one time. Our first bottle neck was the multiple centrifugation steps (Fig. 3). This problem was
overcome by utilising a continuous flow centrifuge acquired for this project, which can easily handle
10 L per hour. The second bottle neck was drying of organic solvents, with the possibility to recycle the solvent. This step
is still somewhat problematic, but we used a combination of medium scale rotary evaporation (0.5-1 L/hour) and spray
drying (2 L/hour) to handle the large volume of organic solvent. Smaller volumes (<500 mL) was reduced by freeze
drying if the solvent composition allowed this procedure. In our original planning for this project all the concentration
steps were to proceed via spray drying, as this method is proposed to handle 2 L per hour, and the solvent can be
collected for recycling. In practice, the nature of some of the liquids did not allow for efficient spray drying and the
recycling of the organic solvents are not so effective. We will have to rethink our strategy and may rather optimise the
rotary evaporation to limit the manual input, as this gives the option of recycling the organic solvent and curb the
chemical waste footprint.
Second, a robust two step methodology was developed to purify the TRCs to >85% purity directly from the organic
extract by exploiting the amphipathicity of the tyrocidines and their analogues (Purification step 2, Fig. 3). Purification
step 2 entails a chromatographic purification which was optimised via a laboratory scale AKTA purification system. With
this system we were able to concentrate the TRCs and remove some of the pigment and most of the GRMs yielding a
>85% pure TRC preparation. This method can directly be up-scaled (50-100 times) on the pilot scale BIOPROCESS
chromatographic system. Unfortunately we were not able to run our large scale crude extracts via the up-scaled
chromatographic system, as the system malfunctioned on several attempts. We were able to get it in working order,
apart from one unit that has failed on the column, which has been on back order for the last three months. What is a
positive aspect from this is that we now have a good knowledge on how this large scale chromatography system
operates and to our knowledge we are currently the only group in South Africa with this technology. As upscaling is the
most difficult step in biotechnology developments, we will be able to complete our own upscaling exercise and assist in
such endeavours in the future.
Esti
mate
d c
ost
in R
an
d
per
gra
m T
CN
pre
para
tio
n
Natural Antimicrobial peptides in Agriculture
Both of the developed purification methodologies are of such a nature as to allow for cost effective, high volume
through-put purification of TCN and TRCs. With the high yields of peptide and the ease of purification, we demonstrated
that it is possible to economically produced large quantities of high value peptide as summarised in Table 3.
Table 3: Summary of tyrothricin production costs to produce 500 g of crude tyrothricin preparation or either of the
two purified preparations. The costing excludes labour, laboratory space costs, general overhead costs, but
includes electricity and instrumentation costs.
Item Crude >75% >85%
Reagents 14535 22229 24990
Fermentation 5134 0 0
Drying 107 107 107
Consumables 1454 1454 2907
Purification 0 200 10800
Analysis 1000 1000 1000
Cost of preparation R 22229 R 24990 R 39803
Cost/g preparation R 44.46 R 49.98 R 79.61
Note that the current market price for >95% TCN at Toku-e is R 21303/g (http://www.toku-
e.com/Tyrothricin-P732.aspx)
The costing summarised in Table 3 provided us with the basis for preliminary financial predictors for commercialisation.
We used the costing to estimate the cost of the TRC and TCN preparations if we up-scaled our production to the
maximum possible scale (2 kg) that the current infrastructure and methodologies can accommodate (Fig. 4).
The maximum production could supply an appreciable number of nurseries with TCN formulation for grafting and micro-
propagation. In our trials we used 1 mg of the crude preparation per woody plant graft, 100 g of the >75% pure TNC
per cutting and only 50 g per micro-propagated plant. For plant cultures we used 150 g/culture of the 85% pure TRCs.
From our prediction we gained very little in cost reduction after 1 kg using the current high cost research grade media
components, with the lowest cost at R 38 per gram of the crude preparation, R 42 per gram for the >75% pure TNC
preparation and R 56 for >85% pure TRCs (Fig 4.). If labour and other overheads are included in the cost, it will at least
quadruple the cost per gram. However, the use of these peptides remains economical, for example the treatment of
1000 cuttings with viable plant value of R 15 will cost only about 4 cents / cutting with a 100% profit margin. A ±1-2%
increase in yield will therefore easily cover the TCN treatment cost.
120
110
100
Crude TCN extract
>75 Pure TRC
>85% Pure TRC
90
80 Maximum in-house
70 production
60
50
40
30 0 1000 2000 3000 4000 5000
Amount of Trc extract (g)
Figure 4: A graphic depiction of the predicted cost per gram of the three different peptide preparations utilising our
optimised production and purification protocol. Calculations are based on the data given in Table 3 and
exclude labour, laboratory space hire and general overheads.
Val-Gramicidin A 6.1
Val-Gramicidin B 0.6
Val-Gramicidin C 0.9
Natural Antimicrobial peptides in Agriculture
Tailoring natural peptide microbicide combinations
The research on tailoring natural peptide microbicide combinations formed part of the PhD thesis of JA Vosloo,
Department of Biochemistry, Stellenbosch University. The TCN production profile, therefore the peptide mixture or active
compounds that are produced, has previously been shown to depend on the concentration of the aromatic amino
acid in the bacterial fermentation medium (Vosloo et al. 2012). On the basis of our experimental data a competitive
binding model was constructed that predicts the amino acid occupancy at the three variable positions as a function of
the concentrations of the aromatic amino acids in the medium. This model can be used to produce tailored peptide
complexes to target specific pathogens via fermentation with a desired amino acid composition (Vosloo, Rautenbach
& Snoep 2015).
Phenylalanine (Phe) supplementation leads to the production of a range of TrcA analogues containing Phe in position 3
and 4; with either tyrosine (Tyr) or Phe at position 7(refer to Fig. 1) (Vosloo et al. 2012). Although TrcA is one of the most
active peptides against fungi, it has a lower antibacterial activity than the other major TRCs (Leussa & Rautenbach,
2014). Furthermore, the production in Phe-containing media is appreciable lower than the normal media C.
Supplementation with tryptophan (Trp) resulted in the predominant production of another group of peptides, the
tryptocidines containing Trp at the variable aromatic position 7 (refer to Fig. 1), but also an increased amount of linear
GRMs. A shift in the production of the different tryptocidine analogues, containing various combinations of Phe and Trp
in positions 3 and 4, occurred when the medium was co-supplemented with high Phe concentrations. The tryptocidines
have good antibacterial activity, but lower antifungal activity (Troskie et al. 2014a), but again the production in Trp
containing media is appreciable lower. Refer to Annexure A for a summary of the antimicrobial activity of the TRC
mixture and TCN.
The combination of peptides in the produced complex is quite important in the formulations. Maximal activity toward
different agronomically relevant fungal pathogens is achieved with peptides with Tyr in position 7 (refer to Fig. 1),
namely the tyrocidines (Troskie et al. 2014a). Therefore for this project we decided to utilise the mixture of peptides
produced with our optimised production protocol in non-supplemented medium C (refer to Table 2). In the medium the
produced peptide complex contains mostly TRCs with Tyr in position 7, in particular the analogues containing Phe in
position 4 namely tyrocidine A (TrcA) and tyrocidine B (TrcB) (Table 4), which have high antifungal and antibacterial
activity respectively (Leussa & Rautenbach, 2014). TrcC with Trp in positions 3 and 4 is the third major peptide and this
peptide has both potent antifungal and antibacterial activity (Leussa & Rautenbach, 2014) (Table 4). The combination
of TrcA and TrcB also showed synergistic activity against fungi (Aspergillus fumigates) and while the combination TrcC
and TrcB showed synergism against bacteria (Bacillus subtilis) (Fig. 5). Other peptide combinations mostly showed
cumulative activity (results not shown).
Table 4: Summary of the chemical composition of TNC75 preparation as determined with UPLC-MS. Refer to Fig. 1 for
more detail on the chemistry of the tyrocidines and analogues.
Component % a
P ig m ent / h ydr op hi li c c o mp o ne nt b 1 6. 4
Tyrocidines and analogues c
Tyrocidine A/A1 21.1/4.4
Tryptocidine A/A1 6.3/0.4
Tyrocidine B/B1 17.9/3.5
Tryptocidine B/B1 6.2/0.5
Tyrocidine C/C1 10.7/1.6
Tryptocidine C/C1 2.5/0.3
Linear gramicidins c
Ile-Gramicidin A 0.6
a The percentage mass contribution of each of the respective components relative to the total b The percentage of the total mass of the fraction collected by semi-preparative HPLC c The percentage contribution of each of the different analogues is expressed in relation to the purity
determined relative to the commercial tyrocidine mixture obtained from the respective UPLC-MS peak
areas of the total TCN and fraction collected by semi-preparative HPLC.
FIC
Trc
A
FIC
Trc
B
Natural Antimicrobial peptides in Agriculture
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1.1 Antifungal activity Antibacterial activity
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
FIC TrcB
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
FIC TrcC
Figure 5: Isobolograms of the combined activity of TrcA and TrcB against the fungal target Aspergillus fumigates, and
TrcC and TrcB against the bacterial target Bacillus subtillis. Fractional inhibition concentrations (FICs) falling
below the red line in the green triangle indicate synergistic activity. Each data point is the mean of 4
determinations with error bars representing the SEM.
Development and testing of formulations of the selected antifungal peptides and combinations
This research on development and testing of formulations of the selected antifungal peptides and combinations formed
part of the PhD thesis of JA Vosloo, Department of Biochemistry, Stellenbosch University. The activity of both TRC and
TCN formulations toward Aspergillus fumigatus has been established, with both the crude >75% and >85% peptide
preparations having good activity. However, both the peptides in the TCN complex and the TRC peptides aggregate at
high concentrations and loose antimicrobial activity in aqueous solutions over time. The high activity of freshly prepared
peptide formulations was maintained when concentrated stocks of these peptide preparations, particularly the crude
TCN, was maintained at solvent concentrations in excess of 75% organic solvent, where ethanol served as the main
solvent. Concentrated stock solutions of these peptide preparations maintained at organic solvent concentrations of
75% (m/v) retained 93±3% of their activity after two months in solution relative to freshly prepared peptide solutions at
the mentioned solvent concentration. Moreover, when the organic solvent concentration was increased to 90% (m/v)
together with the addition of acid, highly concentrated stock solutions of TCN were created. The increased TCN
concentration, as well as the addition of acid thus served to solubilize the peptide formulation and maintain them in
solution.
With the addition of salts that may be present in water used to dilute the stock solutions for field trials, only calcium
chloride had an appreciable effect on the activity of the peptide preparations (Table 5) (Troskie et al. 2014a; Leussa
2014). Calcium in the peptide formulation led to loss of antifungal activity, but the metal-chelating agent,
ethylenediaminetetraacetic acid (EDTA) enhanced the antimicrobial activity of the peptide preparations. However,
we decided not to pursue this formulation option, as EDTA is not classified as a GRAS (generically regarded as safe)
chemical.
Table 5: Influence of additive in TRC formulation on the peptide activity towards the target cell. The peptides were
pre-incubated for 60 minutes in NaCl, KCl, MgCl2 or CaCl2 at concentration ranging from 2.0-100 mM salt.
Microbial target CaCl2 MgCl2 NaCl KCl
Fusarium solani
(fungal target)
Botrytis cinerea
significant decrease at
high concentrations no influence
no
influence
no
slight
decrease
no
(fungal target) decrease no influence influence influence
L. monocytogenes B73
(bacterial target)
significant
increase no influence
no
influence
no
influence
As peptide aggregation leads to loss of activity we assessed a number of organic solvents. Both acetonitrile (ACN) and
dimethylformamide (DMF) as solvent decreased the aggregation of the TRCs and TCN formulation when compared to
ethanol (EtOH) as solvent (results not shown). ACN is a highly toxic solvent and was not considered for formulation. The
improved solubilisation and lower aggregation by DMF corresponded with the observation that DMF increased the
activity of TCN, above that of EtOH as solvent against fungi (Fig. 6).
% A
cti
vit
y
Natural Antimicrobial peptides in Agriculture
130 Aspergillus fumigatis Bacillus subtillis
120
110
100
90
80
70
60
50
40
30
20
10
0
1% DMF
1.5% EtOH
1% DMF
1.5% EtOH
TCN TCN + 5% Suc TCN + 5% Glc
Figure 6: Comparison of the relative activity of TCN75 toward the representative fungal pathogen, Aspergillus
fumigatus and Gram-positive bacterium, Bacillus subtilis. TNC75 was dissolved in either 1.5% (v/v) EtOH or
1.0% (v/v) DMF alone or in the presence of 5% (m/v) of either sucrose (Suc) or glucose (Glc). Inhibition
parameters determined in EtOH was set as 100%. Statistical analysis was done using Bonferroni’s Multiple
comparison test (One Way ANOVA) with P<0.001 when comparing formulations (with and without sugars) in
the two solvents and P<0.01 when comparing the sugar formulations relative to the respective solvents
alone. Each data point is the mean of 3-50 determinations with error bars representing the SEM.
The sugars glucose, sucrose and fructose, except xylitol, significantly decreased (P<0.01) the antimicrobial activity of the
TRC and TCN preparations (Fig. 6). However, the activity of the formulations containing 1% DMF was still sufficient for
general antimicrobial protection (Fig. 6). Although DMF leads to higher activity against fungi it is a harsh industrial solvent
and does not have GRAS status, therefore we decided to only use ethanol for the stock formulations. As ethanol is not
recommended for use with bee feeding trials, the peptide formulations containing sucrose and DMF were used for bee
toxicity studies.
From these results, a number of formulations were prepared for use in our Phase II trials (Table 6).
Table 6: Summary of the TCN and TRC formulations and their applications in this study
Formulation
name
Minimum TRC
content Additives Application
0.2 M HCl,
TCN40A50 40% 75% EtOH Protection of grape grafts
400 fold diluted
0.2 M HCl,
TCN40A40 40% 75% EtOH Protection of peach grafts
500 fold diluted
TCN75A40 TCN75A50
75%
0.2 M HCl,
75% EtOH Micro-propagation of
blackberry cuttings 400/500 fold diluted
Preparation of antimicrobial
TCN75 F4 75% 1.5% EtOH materials; Micro-propagation
of seedlings; Apple cuttings
TCN75 F2 75% 1.5% EtOH Protection of cut flowers
TCN75 F3 75% 1.5% EtOH Protection of cut flowers
TCN75 F2CA 75% 1.5% EtOH, Comm A Protection of cut flowers
TCN75 F3CA 75% 1.5% EtOH, Comm A Protection of cut flowers
TCN75 F2CB 75% 1.5% EtOH, Comm B Protection of cut flowers
TCN75 F3CB 75% 1.5% EtOH, Comm B Protection of cut flowers
TCNGS F2 37% 1.5% EtOH, 25 mg/L GS Protection of cut flowers
TCNGS F3 37% 1.5% EtOH, 35 mg/L GS Protection of cut flowers
Natural Antimicrobial peptides in Agriculture
Table 6: Continued
Formulation
name
Minimum TRC
content Additives Application
0.2 M HCl,
TCN75A F1 75%
TCN75A F4 75%
75% EtOH
1000 fold diluted
0.2 M HCl,
75% EtOH
Sanitation of fruits
Protection of fruits
400 fold diluted
TCN75DS 75% 1% DMF in
50% sucrose
TCN75DC 75% 1% DMF in
Bee toxicity trials
Bee larvae toxicity trials
Diet C
TRC85 F1 85% 1.5% EtOH Protection of plant cultures
TRC Mix >95% 1.5% EtOH Control in purity and
antimicrobial assays
Comm A - commercial biocide A; Comm B - commercial biocide B
Phase II Research results
In vivo evaluation of peptide microbicide formulation toxicity on bees and nematodes
The research on the in vivo evaluation of the toxicity our TCN peptide preparation towards bees formed part of the PhD
thesis of JA Vosloo, Department of Biochemistry, Stellenbosch.
In light of the promising antifungal and antibacterial activity of the TRCs (Troskie et al. 2014; Leussa & Rautenbach, 2014)
and the potential use of TCN and TRC preparations in agriculture, we assessed the eukaryotic cell toxicity and in vivo insect toxicity. The known ~10 mg/L haemolytic activity of highly purified tyrocidines was confirmed, but we found that
our >75% pure extracts containing a yellow-brown pigment from the Br. parabrevis bacterial cultures had a much lower
haemolytic activity at >65 mg/L, which is 5-20 fold higher than the antimicrobial minimum inhibitory concentrations
(MICs). As previously found for eukaryotic cells (Rautenbach et al. 2007) the tyrocidines also showed in vitro toxicity
towards Sf9 insect cells, however, it was less toxic at >20 mg/L (results not shown).
In vivo feeding studies of African honeybees, Apis mellifera scutellata, using TCN75DS formulations showed no toxicity
directly related to the TRCs after the recommended 6 hours, as well as up to 48 hours of feeding in which the individual
bees consuming up to 80 microgram per bee (Fig. 7A). After 2 days of caged feeding no difference in survival was
observed between the bees fed 1.50 g/L TCN75DS relative to the 1% DMF/50% sucrose dosing vehicle control where a
survival >90% was observed (Fig. 7B). Toxicity toward African honeybees was only observed after 72 hours when caged
bees consumed a diet solely composed of sucrose spiked with TCN75 at concentrations of 0.5-1.50 g/L, concentrations
in excess of 30-100 fold the minimum inhibitory concentration (MIC) observed towards the fungal pathogen Aspergillus
fumigatus (Fig. 7B). This toxicity, however, was found to be due of confinement that led to constipation, as field trials in
three hives did not show any toxicity, but rather an improvement in survival (Fig. 8).
After returning the bees to their hives of origin, retrieval of the TCN75DS fed bees was 25-75% greater than the control.
However, this difference was only statistically significant for day 20 (Fig. 8A), although there were major differences
between hives with some hives showing >200% retrieval of bees compared to the untreated controls (Fig. 8B). Weighing
and dissection of bees after caged feeding for 48 hours, as well as those recovered from the hives showed no
discernible difference between the TCN75DS fed bees relative to those of the control (data not shown). Thus, even at
such extremely high concentrations, consumption of TCN by adult bees was safe and may even have been to an
advantage to them.
Am
ou
nt
( g
)
co
nsu
me
d p
er
be
e
% B
ee
re
trie
va
l it
o c
on
tro
l
% Y
ou
ng
bee
mo
rta
lity
% R
etr
ev
al o
f b
ees
ito
co
ntr
ol
Natural Antimicrobial peptides in Agriculture
A 160
140
120
100
80
60
40
20
0
Concentration TCN75DS in bee-feeders
0.05 g/L
0.50 g/L
1.00 g/L
1.50 g/L
B 100
90
80
70
60
50
40
30
20
10
0
1% DMF/50% Suc
0.05 g/L TCN75DS
0.50 g/L
1.00 g/L
1.50 g/L
DiM 35 g/L *
*
* * *
* *
*
0 4 6 24 48 72
Hours of feeding
4 6 24 48 72
Hours of feeding
Figure 7: Consumption of TCN75DS by adult African honey bees relative to the 1% DMF/50% Suc, and DiM controls. A.
Amount of TCN75DS in µg consumed per bee in each of the respective TCN75DS feeding solutions. B. The
relative percentage mortality of bees observed in each of the different feeding solutions over the 72 hour
feeding period corrected relative to the natural mortality in the 50% Suc group using Abbots correction
(Abbot 1925). Statistical analysis was done using Bonferroni’s Multiple comparison test (Two Way ANOVA)
with * P<0.001 relative to 1% DMF/50% Suc. Each data point is the mean of 4 determinations with error bars
representing the SEM.
A 200
P<0.05 B 275 Hive 2a Increase in
bee foraging 175
150
125
100
75
50
25
0
2 5 14 20 28
Days after 1.5 g/L
TCN75DS feeding
250
225
200
175
150
125
100
75
50
25
0
Hive 2b
Hive 3a
Hive 3b
Hive 4a
Hive 4b
0 5 10 15 20 25 30
Days after 1.5 g/L TCN75DS feeding
Figure 8: Comparison of the retrieval of Apis meliffera mature honey bees fed with either control (1% DMF/50% Suc) or
1500 mg/L TCN75DS feeding solutions for 2 days and then returned to their hives of origin. With A showing the
average % retrieval compared to the control and B the retrieval in the respective hives compared to the
untreated controls. Each data point is the mean of 4 determinations with error bars representing the SEM.
Honey bees progress through four different developmental stages between eggs being laid by the queen bee and
incubation (day 1 to 3), they then develop into larvae (day 4 to 9), progress into pre-pupa (day 9-12), pupa (day 13 to
20) before finally emerging as adult bees (day 21) (Jay 1963). As larvae are fed by the adult bees that may have
ingested TCN75, the toxicity of TCN75 needed to be determined towards honey bee larvae. A long term feeding
concentration of 56 mg/L of TCN75DC was found to be the highest concentration the bee larvae could tolerate without
displaying any toxicity (Fig. 9).
% B
ee
la
rva
e m
ort
ality
Natural Antimicrobial peptides in Agriculture
100 #
90
80 #
70
60 #
50
40
30
20
10
0
Day 5
Day 6
Day 7
#
#
*
DiM Vehicle 6.17 18.5 55.6 167 500
mg/L TCN75ES
Treatment
Figure 9: The relative percentage mortality of Apis meliffera honey bee larvae after a single exposure to a range of
concentrations of TCN75DC together with the insecticide DiM at day 4. Statistical analysis was done using
Bonferroni’s Multiple comparison test (One Way ANOVA). The relative mortality after exposure to the vehicle
containing 0.8% DMF was compared to each of the respective treatments at days 5, 6 and 7 # P<0.001;
* P<0.05. Each data point is the mean of 9 determinations with error bars representing the SEM.
The toxicity of a purified TRC, TrcA, was determined as >7.6 mg/L in the long exposure assay towards the nematode
Caenorhabditis elegance (round worm). This concentration of TrcA was used in the successful treatment of Candida
albicans infections in these nematodes (Troskie et al. 2014b). Bee larvae were possibly considerably more resilient to
TCN75ES than these intestinal parasites were to the purified single peptide.
The reduced toxicity of TCN75 toward insect cells supports the very low toxicity of TCN75DS observed toward adult
honey bees. Using concentrations of approximately 200 times the MIC observed toward the representative target
organisms, no influence of the peptide was observed toward the bees fed for the conventional six hours proposed in
the usual toxicity testing. Due to the low solubility of these peptides in water, mortality in adult bees only occurred when
they were exclusively fed TCN75DS for extended time periods. These mortalities occurred at extremely high
concentrations of tyrothricin which would be unattainable by bees foraging within the natural environment. Even if bees
were to be drenched with the peptide solution during application, their low solubility in water would limit the exposure
to minute amounts. Moreover, we found that these peptides adhere very strongly to surfaces due to their amphiphilic
nature (see discussion later) and would therefore further limit any exposure to larvae after application.
It is therefore concluded that the TCN peptides have a very low toxicity toward honey bees and it would be extremely
unlikely to cause an adverse effect toward them when applied within agricultural applications. The fact that the TCN75
formulation also have potent activity again bee pathogens (refer to Annexure A, Table 1A) is also a positive feature of
our peptide formulations, and will probably benefit bee populations that are challenged by high incidence of hive
infections. These results indicated the relative safety of the controlled use of the TCN and the TRCs in a plant protective
role targeting pathogenic fungi in agricultural applications.
In vivo evaluation of peptide microbicide formulations on selected cut flowers
The main aim of the cut flower trials is to determine if treatment with the TCN75 formulation, TCN combined with GS or a
combination of commercial/standard treatment (Comm A and Comm B) will extend the life/commercial viability of cut
flowers. Blue larkspur flower bunches (Delphiniums) were chosen as a model cut flower with a short vase life. Blue
larkspur has less than 3 days vase life with the blue flowers in the flower bunches falling within 2 days. None of the
formulations showed any phytotoxicity and all preserved the flowers better than control treatment where only tap water
was used. The formulations containing both the analogous cyclic peptide GS and the TCN peptides performed as well
as the commercial product, but those containing GS, TCN and the commercial product (Comm A) preserved 50% of
the flower bunches up to day 5 (Fig. 10A). These results indicate that formulations containing TCN have a good potential
in product formulations to extend the vase life of these fragile flowers.
The second flower trial was done on the well-known Gerbera daisies or African daisy, a highly sort after cut-flower with
an average vase life of about a week. In an exploratory trial on Gerberas we found that GS showed phytotoxicity,
therefore focussed on TCN in the formulations for these flowers. All the TCN containing formulations again extended the
vase life of all the Gerbera species, outperforming the commercial product (Comm B) in two of the species (Fig. 11).
Combination of the TCN with Comm B showed a slight improvement above that of Comm B alone, but it was less
Nu
mb
er
of
usa
ble
flo
wers
(n
=1
8)
Nu
mb
er
of
us
ab
le
flo
wer
bu
nc
he
s (
n=
6)
Natural Antimicrobial peptides in Agriculture
effective after day 12 leading to welting (Fig. 11). However, on average the TCN75 F3 extended the vase life of more
than 50% of the flowers to 12 days and out-performed Comm B which is generally used for Gerberas (Fig. 10B).
From the results it was evident that the TCN formulations enhanced the vase life and commercial viability of the flowers
compared to tap water and even a commercial product used as control. After 12 days 55% of the TCN75 F3 treated
Gerberas were still commercially viable, compared to only 17% of the flowers in the no treatment group (tap water) and
22% of flowers treated with the Comm B. Similarly, the addition of TCN supplemented with GS prolonged the life of 50%
of the Delphinium flower bunches to 5 days. The results indicate that TCN formulations are feasible candidates to be
developed as a product for the cut flower industry to extend cut flower life and commercial viability, thereby increasing
the profit margin and incorporating a green economic alternative into this industry.
Delphiniums (Blue larkspur) 6
5
4
3
Vase water treatment
Comm A
TCN75 F2
TCNGS F2CA
TCNGS F3
TCNGS F3CA
Water
2
1
0 2 3 4 5 8
Days in vase
Gerberas (African daisy)
18
16
14
12
10
8
Vase water treatment
Comm B
TCN75 F2
TCN75 F2CB
TCN75 F3
TCN75 F3CB
Water
6
4
2
0 1 6 10 12 16
Days in vase
Figure 10: Effect of vase water treatment on the water uptake of Blue larkspur (Delphinium hybrid) and the African
daisy (Gerbera spp) on the number of usable/saleable flowers over time. Controls contained only tap water,
those with commercial product received the dosage as specified by supplier.
Natural Antimicrobial peptides in Agriculture
Figure 11 Photographic evidence of the influence of the different additives to the vase water of Gerbera mermaid (two
panels on left) and Gerbera larreia (two panels on right) at 12 and 16 days.
In vitro assessment of the surface activity of peptide microbicide formulation
This research on in vitro assessment of the surface activity of peptide microbicide formulations formed part of the MSc
thesis research of W Van Rensburg, Department of Biochemistry, Stellenbosch University.
Agriculture and many other industries experience great losses due to persistent bacterial and fungal infections.
Persistent infections are attributed to antibiotic or biocide resistance, mostly because of the formation of biofilms. Since
the treatment of biofilms is problematic, prevention of microorganism colonization to the surface can be done by
modification of solid surfaces by covalent coupling coating or absorption of antimicrobial agents. The cyclic TRC
peptides also have an inherent bio-stability and tend to adhere to both hydrophilic and hydrophobic surfaces, making
them ideal candidates to develop antimicrobial surfaces.
A variety of natural, semisynthetic and synthetic materials were treated with TCN75 F4 and subsequently analysed for
material character and antimicrobial activity (Table 7). Peptide desorption and subsequent analysis by mass
spectroscopy was successfully used to confirm the presence and integrity of the TRCs adsorbed (results not shown).
Scanning electron microscopy showed that the adsorbed peptides did not affect the structural integrity of the treated
materials (results no shown). However, it was shown that the adsorbed peptides changed the hydrophobic/hydrophilic
character by means of a wettability assay of some materials (Table 7).
Natural Antimicrobial peptides in Agriculture
Table 7: Summary of the characteristics and conventional use of selected filters utilised in this study and the influence
of treatment with TCN75 F4.
Material
Type
Monomer structure(s)
Conventional use
Influence of
TCN75 F4 treatment on material
Polycarbonate
(PC)
Synthetic
polymer
Solvent filtration, various
industrial applications,
plastic ware
No character
change
Polypropylene
(PP)
Synthetic
polymer
Various industrial&
medical applications,
plastic ware
Increase in
hydrophobicity,
antibacterial
Polyvinylidene
difluoride (PVDF)
Synthetic
polymer
Solvent filtration, various
industrial applications,
plastic ware
Increase in
hydrophobicity,
antibacterial
Polystyrene
Synthetic
polymer
Various industrial &
medical applications,
plastic ware, glass
substitute
Character change
unknown
Poly(methyl
methacrylate
(PMM)
Synthetic
polymer
Various industrial &
medical applications,
plastic ware, glass
substitute
Character change
unknown,
antibacterial
3% Chitin spun
on PMM fibres
(CH-PMM)
Semi-
synthetic
nanofibres
Chitin
Filtration, various novel
applications, flexible
materials
Character change
unknown,
antibacterial
3% Chitin spun
on cellulose
whiskers
(CH-CL)
Natural
nanofibres
Filtration, various novel
applications, flexible
materials
Character change
unknown,
antibacterial
High density
cellulose (HDC)
Natural
polymer
Packaging, paper
products
No character
change,
antibacterial
Cellulose (CL)
Natural
polymer
Filtration, packaging,
paper products, wood
products
No character
change,
antimicrobial
Mixed cellulose
ester – cellulose
acetate and
nitrocellulose
(NCCA)
Semi-
synthetic
polymer
Solvent filtration, various
industrial applications
Increase in
hydrophobicity,
antibacterial
Cellulose
acetate (CA)
Semi-
synthetic
polymer
Sterile filtration, various
industrial applications,
plastic ware
Decrease in
hydrophobicity,
slightly antibacterial
We tested the solid phase activity of TCN treated materials against the model Gram-positive bacterium Micrococcus
luteus using assays developed to test solid phase antibacterial activity in a low nutrient environment. Five of the ten
tyrocidine treated materials namely: CL, HDC, PMM and the two chitin containing nanofibres showed a >80% sterilisation
capacity against a very high bacterial count of >104 Micrococcus luteus cells on the 5 mm disks (Fig. 12).
% A
nti
bat
eria
l act
ivit
y
% B
ac
teri
al in
hib
itio
n
% A
nti
bac
teri
al a
ctiv
ity
Natural Antimicrobial peptides in Agriculture
120 Synthetic materials
Natural materials
100 Semi-synthetic materials
80
60
40
20
0
Type of material
Figure 12: Comparison of retained antimicrobial activity of different materials treated with TCN75 F4. The inhibitory
activity was determined in a low nutrient environment with high bacterial cell count (7x104 Micrococcus
luteus cells/well or 5 mm filter disk) using the Alamar Blue viability assay. Bars represent the average of 6-9
determinations with SEM.
Treated CA and NCCA, PP and PS exhibited low activity, while treated PC could not inhibit the high cell numbers of
Micrococcus luteus. However, the TCN treated PC did show an appreciable activity with lower cell numbers in a high
nutrient environment (results not shown). TCN75 F4 treated CL filters (5 mm in diameter) showed 100% activity against 105
cells of the food pathogen Listeria monocytogenes and Micrococcus luteus (results not shown). Refer to Annexure A
tables 1A & 2A for data on the broad-spectrum TCN and TRC antibacterial activity.
Stability of the antimicrobial activity was also further tested by filtering various solvents through the CL filters (used as
paper substitute), such as 2% salt (NaCl) and organic solvent (50% and 100% acetonitrile). Only the 50% organic solvent
led to a decrease in activity, but the remaining activity was still >50% (Fig. 13A). The retained activity against bacteria is
also reasonably stable over time, but only decline to about 60% potency after 18 months due to natural degradation
and possibly the Maillard reaction with polysaccharide containing materials such as cellulose (paper and wood) (Fig.
13B).
A 110
100
90
80
70
60
50
40
30
20
10
0
*** ***
B 100
90
80
70
60
50
40
30
20
10
0
***
***
Wash solution
Figure 13: The effect of washing of CL filters treated with TCN75 F4 with different solvents on the sterility of the filters as
determined with a vitality assay with Micrococcus luteus as bacterial contaminant (A). Each data point
represents the mean of at least 24 determinations with SEM. The graph on B shows the retention of activity
over time on the antibacterial activity of TCN75 F4 treated CL. Each data point represents the mean of 6-30
determinations with SEM. Statistical analyses in A and B were done using Bonferroni’s Multiple comparison
test (One Way ANOVA) with *** P<0.001. Bars represent the average of 6-9 determinations with SEM.
Natural Antimicrobial peptides in Agriculture
The TCN treated CL filters were also tested for antimicrobial stability at extreme temperatures and pH, as well as after
multiple water washes. It was found that the CL filters maintained their antimicrobial activity after 12 water washes, after
heating to 100oC and after being treated between pH 1-10 (results not shown). At pH >10, in particular pH 13, the filter
lost some antimicrobial activity most probably due to the degradation of the cellulose (results not shown).
In the light that cellulose retained good antimicrobial activity wood cuttings (grape vine stalks) were treated with a
solution containing 10 and 20 mg/L TCN75 for the inhibition of three of the major fungal pathogens of grape vine. We
found that 20 mg/mL TCN75 where sufficient to kill 2000 spores/mL of these pathogens (Table 8). This result indicated
that the TCN preparation could be used to protect grape grafts from fungal pathogens. Refer to Annexure A Table 3A
for data on the broad-spectrum TCN and TRC antifungal activity.
Table 8: The “in vivo” antifungal activity of TCN75 in the presence and absence of grapevine stalk material as
determined with a viability assay. The percentage viable spores compared to the control are indicated
(data courtesy of Dr A de Beer).
Cylindrocarpon Phomopsis Phaeoacremonium
Concentration - wood + wood - wood + wood - wood + wood
10 mg/L 0 40 0 0 0 8
20 mg/mL 0 0 nd nd 0 0
TCN maintains its antimicrobial activity when adsorbed to a variety of natural, semi-synthetic and synthetic materials. It
can therefore be concluded that TCN treated solid surfaces holds great potential in preventing the initial microbial
colonization and subsequent contamination and biofilm formation. It is hypothesised that TRCs prefer to bind to
hydrophilic surfaces exposing the hydrophobic residues and the cationic residue of the peptide to interact with the
bacterial or fungal surface in order to elicit an antimicrobial response. The TRCs showed a preference for adsorption
onto cellulose and cellulose analogues, as well as wood cuttings which points to possible application in protective food
wrapping and wood surface protection. Furthermore the TRCs retain long term activity when adsorbed to solid matrixes
and is very stable regardless of multiple washes, extreme pH exposure and boiling. Accordingly TRC treated materials, in
particular containing cellulose can be developed and tailored to a specific application, such as in air/water filters and
packaging/wrapping materials used for processed food, fruits and vegetables.
In vivo evaluation of peptide microbicide formulations on selected seasonal fruits
With the results that the TRCs associated with a number of natural and synthetic materials and retains it antimicrobial
activity on the surfaces, we assessed the sterilisation potential of seasonal fruits. A small trial on oranges, the model
winter fruit, was done in collaboration with ICA. Standardised sanitation/protective tests were performed on oranges
using Penicilium digitatum (green mould) and Geothrichum citri-aurantii (sour rot) as target organisms and result are
given in Table 9. We included the cyclic peptide GS in this study as this peptide has similar activity to that of the
analogous TRC cyclic peptides. From these results it is clear that GS performed better than TCN against green mould.
However, neither treatment regime offered full protection or sanitation, whereas the chemical biocide (Comm C at
much higher concentrations) did lead to near 100% protection. The reason for the lower protection than Comm C is
because we limited the dosage or TCN75 to 20 and 50 mg/L and the fungal loads used in the sanitation and protection
trials are very high (>5000 spores per mL). This not representative of environmental spore loads that are normally 10-1000
spores per mL and we may be underestimating the effect of our peptides in produce sanitation protection.
Trials on strawberries (model summer fruit) were unsuccessful, as very little protection against Botrytis cinerea infections
was found with the treatment regime. We will have to rethink both the treatment formulation and regime in order to get
the benefit of the high antifungal activity of the TRCs (Troskie et al. 2014a, Annexure Table 3A).
nd
Ro
ot L
eng
th (m
m)
To
tal p
lan
t bio
mas
s (m
g)
Natural Antimicrobial peptides in Agriculture
Table 9: Summary of orange treatment (sanitation/protection) trial with natural antimicrobial peptide preparations
(data courtesy Dr W. Schreuder from ICA).
Treatment type Peptide Formulation Green mould Sour rot
Sanitation 20 g/L TCN75A
30% decrease in
spoilage
20 g/L GS 70% decrease in
50% decrease
in spoilage
Protection of
50 g/L TCN75A
spoilage nd
57% decrease in
wound infection nd
wounded fruits 50 g/L GS
80% decrease in
wound infection
We delayed further studies on fruit protection as we have recently signed a NDA with BioCHOS, a spin-out company
from the Norwegian University of Life Sciences to work on a formulation containing our peptides for fresh produce
protection.
In vivo evaluation of peptide microbicide formulations in plant cultures
An exploratory applied study on the effect of TCN75 F4 treated CL filters on the sterilization, germination and effect on
tomato seedlings was conducted. It was found that TCN had no effect on the germination. It fully sterilised the filters
against bacterial contamination, but only offered partial protection against fungal contamination. Some phytotoxicity
was found for the filters threated with TCN75 F4 that led to shorter roots and a slight decrease in plant biomass (Fig. 14).
However, the CL filters treated with 5 mg/L TCN75 showed a significant stimulation of root growth (Fig. 14) which
correlated very well with the promotion of root growth we found with our grape plant culture studies (refer to Fig. 9).
A 110
100
90
80
70
60
50
40
30
20
10
0
*** ***
*** ***
***
B *** 50
40
30
20
10
0
Control 5 mg/L 25 mg/L 50 mg/L
[TCN] of filter treatment
Control 5 mg/L 25 mg/L 50 mg/L
[TCN] of filter treatment
Figure 14: The effect of CL filters treated at varying concentrations of TCN75 (0, 5, 25 and 50 mg/L) on root length (A)
and total biomass of tomato seeds after germination (B) on the filters. For each filter treatment the
germinated plants of 75-100 seeds were analysed. Statistical analysis was done using Bonferroni’s Multiple
comparison test (One Way ANOVA) with control compared to TNC75 treatments with *** P<0.001. Bars
represent the average of 40-95 determinations with SEM.
In a trial on a model woody plant, Vitis vinifera (grapevine), the effect of the tyrocidines on the growth of cuttings was
assessed using tissue culture medium supplemented with the TRC85 F1. The progress of plant growth, foliage and root
development was monitored for approximately two months and the growth evaluated visually in terms of survival, root
formation and number of leafs (Fig. 15).
% P
lan
ts (
n=
12 p
er
tre
atm
en
t)
Natural Antimicrobial peptides in Agriculture
110
100 * *
90
80
70
60
50
40
30
20
10
0
***
Medium additive Water
TRC85 F1
*
**
Plant growth and progression
Figure 15: The influence of TRC85 F1 on the vitality and growth of Vitis vinifera (grapevine) cuttings over two months.
The bar graph shows the comparison of TRC85 F1 supplementation with control media of growth parameters
over two months, with photographic evidence on a selection of cultivars after two months. Statistical analysis
was done using Bonferroni’s Multiple comparison test (One Way ANOVA) with control compared to TRC85 F1
treatment with * P<0.0%; ** P<0.01; *** P<0.001. Bars represent the average of 12 determinations with SEM.
After two months 91% viable explants was observed for the TRC treated cultures, which was significantly higher than the
58% of the untreated control cultures (Fig. 15). Significant difference between growth, especially root growth of the TRC
treated and untreated plants were also observed. The treated plants had a higher incidence of root growth (73% vs
25%) and the roots that formed were longer than the roots of the control plants (Fig. 15). The average leaf formation of
the peptide treated plants (3.5±0.4 per plant) was also significantly higher than that of the control plants (1.8±0.5 per
plant). These results are highly promising and warrant further research into the growth enhancing activity of the
tyrocidines for plant cultures.
% P
lan
ts (
n=
16
per
treatm
en
t)
Natural Antimicrobial peptides in Agriculture With the promising results for grape vine plant cultures as a woody plant model, we followed it up by assessing the
influence of TNC75 F4 on the vitality and growth of micro-propagated seedlings of Arabidopsis thaliana (African violet)
as the non-woody plant model (Fig. 16).
100 *
90
80
70
60
50
40
30
20
10
0
Soil additive Water
TCN75 F4
*
Gernination Roots Mature Leafs
Plant growth and progression
Figure 16: The influence of TNC75 F4 on the vitality and growth of Arabidopsis thaliana micro-propagated seedlings
over 5 weeks. The bar graph shows the comparison with control media of growth parameters, with
photographic evidence on a selection of cultivars after two months. Statistical analysis was done using
Bonferroni’s Multiple comparison test (One Way ANOVA) with control compared to TNC75 F4 treatment at
with * P<0.05. Bars represent the average of 16 determinations with SEM.
Natural Antimicrobial peptides in Agriculture
Treatment of the Arabidopsis thaliana seeds and seedlings with TCN75 F4 led to significantly higher germination and
improved growth, particularly leading to significantly more mature leafs (Fig. 16). We did not observe any toxicity
towards these plants and the plants looked visibly healthier that the controls (refer to photographs in Fig. 16), indicating
that there was phytostimulation and that the soil fertility was maintained. Our in vitro activity data against a few soil
organisms did indicate that they are more resistant than many of the bacterial pathogens, therefore these beneficial
bacteria probably survive the TCN treatment and help to maintain the soil fertility (refer to Annexure A, Table 1A). This
corroborated our studies on Vitis vinifera in plant cultures and show that less hardy non-woody plants can also be
treated with TRC and TCN preparations.
In vivo evaluation of peptide microbicide formulation on woody plant grafts and cuttings
The major agricultural industries in the Western Cape, namely the fruit and wine/spirits industries depend on woody
plants such as grape vines, fruit trees and berry plants. The fruit bearing plants are either propagated via grating or
cuttings. These types of propagation are labour intensive and are done in specialised nurseries that produce millions of
plants per season (Fig. 17). However, losses due to graft and cutting failures can vary from only a few percent to near
100%, with some of the major failures caused by fungal infections (Table. 10).
Figure 17: The process of grape vine grafting at Fleury Nursery in Wellington with grafting process of the grape cultivars
to a robust root cultivar performed by skilled artisans (A); waxed grape vine grafts after treatment in wood
pallets (B); grafts covered with wood shavings for the 1-2 months incubation period (C); counting of young
geminated grape vine plants in vineyard after 3 months (D), harvesting of grape vine plants after 10-11
months (E); sorting and grading of young vines (F); storage of viable young plants (G) and grape vine plant
bundles ready for delivery to farmers (H).
% G
rap
e P
lan
ts
Natural Antimicrobial peptides in Agriculture Our results on plant cultures and seedlings showed that the TCN formulations could both protect plants and act as
phyto-stimulator in an agricultural setting. A small field trial with 2250 Merbien grape vine grafts treated with our TCN40A
F4 peptide formulation in the 2013/14 season yielded similar results than the controls treated with conventional chemical
biocide (Comm C) (Fig. 18, Table 10). Treatment with both the TCN40A F4 and Comm C, however, showed lower yields
that indicated that the combination with the chemical biocide may be toxic for the plants (Table 10).
50 75*1*9
Control Treatment Comm C
TNC40A50 treatment
55000
2250
40 7536
30 21300
30000
20
10
0
Germinated (2014/15) Harvested (2014/15) Harvested (2013/14)
Figure 18: The influence of TNC40A50 on the germination and growth of Merbien grapevine grafts after three months
and the harvested Class 1 yields. The bar graph shows the comparison with control treatments. The number
of grafts in each trial is indicated above each bar. The photographic evidence of the germinated plants in
the vineyard is shown after two months. Statistical analysis was done using Students test with control Comm
C treatment compared to TNC40A50 treatment with ** P<0.01.
A large field trial with 32000 TCN treated grape grafts and 28000 grafts treated with Comm C were done in the 2014/15
season. Assessment of the germination the young grape plants after three months of growth in the vineyard indicated
that there was a significantly higher germination yield of 9% (P<0.01) for the TCN40A F4 treated group (Fig. 18). The
increase in grape vine graft germination yields in an agricultural setting correlated with the positive effect of our
TCN/TRC treatments with plant cultures and micro-propagated plants. However, the 2014/15 season were unfortunately
stricken with a drought with only 13 mm rain from January to March 2015 versus 216 mm in the same period in 2014.
(2 different cuttings) 500 2.0
Rosenhof, Late season peach grafts TCN40A40 2014/15 1003 2
Ceres Comm E 1036 2
Rosenhof, In season peach TCN40A40 2015/16 10000 Pending (Jan 2016)
Ceres grafts Comm E 10000 Pending (Jan 2016)
Rosenhof, In season blueberry TCN75A40 2014/15 232 88
Ceres cuttings, micro-
propagation Water 268 77
Rosenhof, Late season blueberry TCN75A50 2015/16 810 Pending (Feb 2016)
Ceres cuttings, micro- Water 810 Pending (Feb 2016)
Natural Antimicrobial peptides in Agriculture
As expected, the viable plant yields for all treatments in the 2014/15 season were significantly lower than the 2013/14
season (Fig. 18, Table 10). Our treated plants generally had more foliage than the slower growing control plants (refer to
photographs in Fig. 18) which is possibly the reason why they were more vulnerable to the drought in these very hot
summer months. Therefore the advantage of higher viable plant yields was lost and the yield was lower, but still
reasonably comparable with the control Comm C treatment (Fig. 18, Table 10). This result was disappointing, but did
indicate that the agricultural practice could be adapted to protect the faster growing plants against such drought
induced decline to retain the higher yields that were indicated by the significantly improved germination (Fig. 18, Table
10).
Table 10: Summary of completed and ongoing nursery trials in the Western Cape
Nursery Detail of trial Treatment
Season Number of
plants
%Yield
Fleury
In season Merbien
TCN40A50
2013/14 55000
na (48)*
Nursery, grapevine grafts
Wellington Comm C 2250 54 (46)*
TCN40A50 + Comm C 2250 46 (41)*
Fleury In season Merbien TCN40A50 2014/15 30000 30 (25)*
Nursery,
Wellington
grapevine grafts Comm C 21300 35 (30)*
Stragrow In season Flame TCN75A F4 2015/16 500 17
Nursery, grapevine grafts (2 different graft 500 18
Citrusdal treatments) Treatment of sawdust 500 3
Comm C 500 na
Stargrow In season apple TCN75A F4 2015/16 500 0.2
Nursery
Citrusdal
M7 hard cuttings (2 different cuttings)
TCN75A F4 + Comm D
(2 different cuttings)
Comm D
500
500
500
500
0.2
8.4
4.2
5.6
propagation
* Class 1 yield is shown in brackets
We conducted a number of other trials at two other larger nurseries with our TCN formulations (Table 10). A late season
peach graft trial in collaboration with Rosenhof nursery gave low yields for all treatments, probably because the grafting
was done too late in the season. However, no toxicity of our TCN formulations was observed and no difference between
TCN40A F4 and commercial biocide treatments was found. The results for a much larger 2015/16 season trial with
peaches are pending. We also conducted a 2014/15 season trial on blackberry cuttings which showed an 11% higher
yield in of viable plants, indicating the possible application of our treatments for micro-propagation of plant cuttings in
nurseries (Table 10). This was followed by a 2015/2016 trial on blackberries and the results are pending. We recently also
endeavoured on a number of other field trials, in collaboration with one of the prominent farming nurseries, Stargrow
Nursery (Table 10). The combination of our TCN formulation with Comm D almost doubled the yield of young apple
trees although it did not work well on its own with a single treatment. For the direct treatment of grape grafts the TRC
formulations also led to a marked improvement of yields versus no graft treatment, with only the a sawdust treatment.
Natural Antimicrobial peptides in Agriculture
8. CONCLUSIONS
8.1 General conclusions Most of the biocides used in industry and agriculture have a number of environmentally harmful effects, and cannot
truly be regarded as “green” chemicals. Chemicals that harm the environment will eventually lead to loss of
sustainability and even if the use these chemicals are cost effective in the short term, a high cost of rehabilitating the
environment to regain sustainability may lead to a total shut down of the industry or agricultural endeavour. With the
movement towards a green economy and eco-friendly environmental practices, there is a growing need for natural
products, which we could address with our natural antimicrobial peptide products.
Advantages of our antimicrobial peptides and formulations
Our natural TCN derived antimicrobial peptide preparations:
are bio-degradable natural compounds leaving only breakdown products with nutritional value for plants and
beneficial microorganisms in the environment;
have low oral and surface toxicity towards mammals, bees and nematodes;
have broad spectrum activity including antifungal, antibacterial and antiviral activity;
have the potential to combat various pathogens, including plant fungal pathogens to which no fully effective
fungicide is available;
show plant growth promoting activity and may stimulate plant resistance;
have a broad spectrum of potential applications including as microbial ecosystem modulators, root protectors,
plant growth promoters and pathogen-free plant propagation;
can be used as antimicrobial/antifungal agents for surface, emulsion, culture media and solution sterilization;
are cost effective with high levels of both crude and fine chemical grade production;
contain rare compounds that are of research interest;
are supported by high level research and development;
are patented and production has been kept as a trade secret.
Impact on Agriculture
This is the first study to show that natural antimicrobial peptides such as the tyrocidines have immense potential to
improve the propagation of plants from seeds, grafts and cuttings for organic and natural farmers, as well as nurseries in
general, thus supporting sustainable farming. Healthier plants which are more disease resistant will lead to higher yields
and would lead to a decrease in environmentally damaging agricultural practices using chemical biocides and
inorganic fertilisers. The agricultural produce can also be protected by using the natural peptide microbicides, leading
to higher post-harvest yields. We were also able to show that our tyrocidine formulations offered some protection
against the economic losses due to spoilage of produce, for example it curbed the spoilage of citrus fruits, as well as
significantly extending the vase life of selected cut-flowers.
Impact on Industry
Natural antimicrobial peptide products, such as the tyrocidine treated cellulose could find application in air and water
filtration, the paper/packaging industries, food and beverage industries, as well as in cosmetic and skin health industries.
The cellulose type of wrapping/packaging containing the TRCs will be fully biodegradable to nutrients that can be used
by plants and beneficial soil organisms, which make the combination of TRCs with cellulose the ideal combination for
biodegradable packaging material for fruits and vegetables. It could replace some of the harmful biocides or products
and will not only have positive impact on the environment, but also have a positive impact on human health.
Natural Antimicrobial peptides in Agriculture
8.2 Key Policy Messages
Improving public knowledge, attitudes, skills, and abilities
The majority of current agricultural sector practices are focussed on production and not generally concerned with
environmental issues or chemical footprints. Although there is a public demand for chemical-free produce, the general
public still prefer to buy the produce that visibly look healthier and that are less expensive, without really thinking about
the chemical impact of produce production. The lower produce price and healthier appearance normally comes with
a high unnatural chemical footprint, namely multiple pre- and post-harvest additives, biocide and pesticide treatments.
Neither the farming practice of maximum production nor the consumer’s preferences for the best produce at the
cheapest price will realistically change within the foreseen future. The only way to lower the unnatural chemical
footprint is if natural alternatives with similar or better produce outcomes and prices come on to the market or if the
harmful unnatural chemicals are banned and the agricultural sector is forced to replace it with less harmful alternatives.
However, there is a lesson to be learned from the wide spread use of the insecticide DDT (dichloro-
diphenyltrichloroethane) and replacement after banning with insecticides that also have a long term environmental
impact such as killing beneficial insects such as bees.
Sensitising the agricultural sector and the public to the possibility of natural alternatives in bio-control is crucial, although
changing dogmatic perspectives will not be easy. Education on aspects of the natural production of food needs to
start as early as secondary school and must preferably be included in tertiary/higher education curricula. The principal
investigator of this project, Prof Marina Rautenbach, received the prestigious award of South African Distinguished
Woman Scientist (Physical and Engineering Sciences) (SA-DWISE) from the South African Department of Science and
Technology (DST) in August 2014. As she is the first woman at Stellenbosch University to receive this award since its
inception in 2003, Stellenbosch University and the DST have showcased her career and research, focussing on the great
potential of antimicrobial peptides that is supported by the Green Fund project. This SA-DWISE award was also the ideal
vehicle to improve the public knowledge on antimicrobial peptides, as was done with a variety of interviews with, and
presentations and lectures by Prof Rautenbach. A concerted effort has been and will still be made to promote this work
and the positive aspects of natural bio-control.
Changing practices, decision making, policies (including regulatory policies), social actions
Translational research is not trivial and we are entering primarily unchartered territories with different rules to that of
basic research. We have been and are still conducting trials at large nurseries in the vineyard- and fruit centres of the
Boland (Wellington, Ceres and Citrusdal). We also had a number of requests further afield by nurseries and farmers that
are willing to participate in our trials. From our interactions with farmers, farming nurseries and nurseries we know there is
a major need for biocide alternatives and we are entering this area at the right time to change agricultural practices.
The positive results for producing more and healthier plants could lead these large nurseries to change their practice of
primarily using chemical biocides and will certainly influence other smaller nurseries to follow suit. Healthier planting
material going to farmers will also lead to lowering the use of harmful chemicals. However, the practice of using harmful
and toxic biocide will not be easily abandoned as there are dogmatic believes that certain unnatural chemical
biocides and products are imperative for good crop yields. In many cases certain chemical biocides are used as it is
the norm, as there is a fear of crop losses if a more natural approach is followed. The only way to change such practice
is to show that natural products such as our natural antimicrobial peptides are viable cost-effective alternatives to the
unnatural chemical biocides. This can only be accomplished by completing successful field trials with natural peptides
and providing such natural products at an affordable price to the nurseries.
Improving social, economic, civic, or environmental conditions
The improvement of environmental conditions, namely helping to regain the natural microbial and plant interactions by
lowering the use of toxic chemicals is the main long term goal of introducing natural antimicrobial peptides as
alternatives for toxic chemical biocides in agriculture and in the industry.
8.3 Recommendations for Further Research / Action The primary goal of this study was to obtain “proof of concept” data for the application of natural TCN based peptide
microbicides in agriculture, in particular for plant propagation and protection of produce. From the results we are
confident that we have the first proof to develop our concept of natural peptide microbicides, such as the TRCs, as well
as other natural antimicrobial peptides that we have recently discovered in a parallel soil bio-mining project, into
agricultural and industrial applications. Concerning the phase II research of this project, we have registered an EU
patent, our USA patent registration is pending and a second patent is in PCT phase.
We are currently the only group in South Africa (and Africa), to our knowledge, working with novel groups of natural
cyclic peptide microbicides and applying it in agricultural research. From the positive outcomes of this research, our
collaboration with the nurseries will continue with exploratory field trials on different plants of high economic value.
Natural Antimicrobial peptides in Agriculture
In order to continue with these trials we need to improve the medium scale production of the TRCs, as well as develop
production strategies for the new peptides candidates that we have recently discovered.
The study on plant cultures and micro-propagation will be extended, focusing on the influence of our peptide
formulations on phyto-stimulation and ISR. Although we envision the use of the TCN formulations only in controlled plant
biotechnological and nursery applications, these peptides are still biocides and all biocides have some toxicity.
Therefore, we will further investigate the bio-stability and bio-degradability of our peptide formulations, as well as their
long term effect on soil fertility and beneficial soil-bacteria. We will also endeavour into industrial trials with our
antimicrobial materials and are going to further exploit the use of our formulations in the cut-flower industry. This
extended long term project will enable us to build a niche in natural peptide microbicide research and hopefully help
to change and curb the over-utilisation of harmful unnatural biocides in South Africa.
A secondary goal of this project was the up-scaling of peptide production and isolation of the highly active peptides
that forms the basis of the natural peptide microbicide formulations. The up-scaled production and purification data
form the basis for developing economical pilot and large scale industrial production protocols that we hope to develop
further with a suitable, preferably South African, industrial partner. Our production and isolation methodology can also
be used to produce purified peptides for fine chemical market, placing us on track for biotechnological advance that
will benefit the South African biotechnological focus.
The long term goal of the extended project is to develop a Biotechnological business around the economic production
environmentally friendly biodegradable microbicides, based on naturally produced peptides. The tailored natural
peptide microbicide formulations could be applied to floriculture, agriculture and biotechnical industries to prevent and
control microbial pathogen carry-over to plants/products, promote plant growth and health and protect against
microbial infections in formulated/stored/transported products and industrial environments. As shown in this exploratory
project, these green peptide microbicides can be utilised to protect plants from the seed, grafts, cuttings or tissue
culture phase through to the established young plants in nurseries and final products in the market. The encouraging
results that we obtained during could lead to products that have a positive influence on the production of healthy
planting material and will hopefully aid in the future to increase the “greener” production of marketable plant products
in South Africa, Africa and possibly worldwide. The agricultural sector, particularly the farmers and nurseries, as well as
the consumer could certainly benefit, as healthy planting material needs less chemical treatments and increase the
changes for better production and thus sustainable farming.
AKNOWLEDGEMENTS
We want to acknowledge the Department of Biochemistry, Stellenbosch University for supplying the laboratory space
for our newly acquired instrumentation, as well as all the support staff helping us to maintain the facilities. Our sincere
thanks to the skilled artisans who did the plant grafting and micro-propagations, maintained the plants and graded the
final products from our trials at the nurseries. We are also indebted to all our collaborators who supplied us with valuable
scientific advice and interactions, allowed us to work in their facilities, use their instruments or conducted extensive
experiments to test and analyse our peptides and formulations.
Natural Antimicrobial peptides in Agriculture
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Natural Antimicrobial peptides in Agriculture
ANNEXURE A Broad spectrum antibacterial activity of the TRCs and TCN against Gram-positive bacterial strains
Table 1A: In vitro antimicrobial activity of TRC Mix and TRC85* against a selection of Gram positive bacterial species.
Concentrations that give minimum inhibition concentration (MIC) are given in µg/mL.
Bacillus subtilis
Organism MIC (µg/mL) Comment
168
OKB105
11/13*
50
Model soil organism
GM B. subtillis 168, surfactin producer
OKB120 20 GM B. subtillis 105
ATCC21332
LMG 2099
f.sp. spizizenii ATCC 6633
50
21*
21
Soil isolate, surfactin producer
Bee gut isolate/soil organism
Soil/marine organism
Bacillus megaterium LMG 7127
Bacillus pumilus LMG 3455
19*
15*
Bee gut isolate/soil organism
Bee gut isolate/soil/bio-control organism
Brevibacillus borstelensis DSM 6347 >100* Bee gut isolate/environmental organism
Enterococcus faecalis DSM 20376 8* Bee gut/isolate/pathogen/gut commensal
Listeria monocytogenes B73 23 Meat isolate/food pathogen
Listeria monocytogenes B73-MR1 14 Meat isolate/ food pathogen, LCN A resistant
Melissococcus plutonius LMG 20360 13* Bee pathogen
Micrococcus luteus NCTC 8340 6 Model Gram+ environmental organism
Paenibacillus larvae
ERIC I, DSM 7030
6*
Bee pathogen
ERIC II, DSM 25430
ERIC III, LMG 16252
ERIC IV, LMG 16247
ERIC I, Isolate 11
ERIC I, Isolate 15
ERIC I, Isolate 24
ERIC I, Isolate 25
ERIC I, Isolate 138
ERIC I, Isolate 145
ERIC II, Isolate 1
ERIC II, Isolate 3
ERIC II, Isolate 6
ERIC II, Isolate 7
ERIC II, Isolate 17
3*
2*
7*
1*
<<0.8*
<<0.8*
29*
<<0.8*
22*
25*
3*
1*
3*
<<0.8*
Bee pathogen
Bee pathogen
Bee pathogen
Bee pathogen
Bee pathogen
Bee pathogen
Bee pathogen
Bee pathogen
Bee pathogen
Bee pathogen
Bee pathogen
Bee pathogen
Bee pathogen
Bee pathogen
Paenibacillus alvei DSM 29* 2* Bee pathogen
Planococcus maritimus DSM 17275* 16* Bee pathogen
Staphylococcus pasteuri DSM 30868* 10* Bee gut isolate/pathogen
Streptomyces griseus DSM 1471* 10* Bee gut isolate/soil organism
Natural Antimicrobial peptides in Agriculture
Antibacterial activity of the TRCs and TCN against Gram-negative bacterial strains Table 2A: In vitro antimicrobial activity of TRC Mix and TCN75* against a selection of Gram positive bacterial species.
Concentrations that give minimum inhibition concentration (MIC) are given in µg/mL.
Organism MIC (µg/mL) Comment
Escherichia coli HB101 >100 Model Gram - organism
Comamonas denitrificans LMG 21602* 48* Bee gut isolate/denitrifying organism
Delftia acidovorans LMG 1226* >100* Bee gut isolate/emerging human
pathogen
Gluconobacter oxydans DSM 2003* >100* Bee gut isolate/acetic acid producer
Janthinobacterium lividum LMG 2892* >100* Bee gut isolate/soil organism
Pedobacter africanus LMG 10345* 77* Bee gut isolate/heparinise producer
Planomicrobium okeanokoites DSM 15489* >100* Bee gut isolate/soil organism
Pseudomonas fluorescens DSM 6147* >100* Bee gut isolate/plant pathogen
Ralstonia picketti LMG 5342* .>100* Bee gut isolate/soil organism
Saccharibacter floricola LMG 23170* 19* Bee gut isolate/ acetic acid producer
Salmonella enterica DSM 11320* >100* Bee gut isolate /Food pathogen
Natural Antimicrobial peptides in Agriculture
Broad spectrum antifungal activity of the TRCs and TCN against fungal pathogens Table 3A: In vitro antimicrobial activity of TRC Mix and TRC85 (*data courtesy Janssen’s Pharmaceuticals) against a
selection of fungal species. Concentrations that give minimum inhibition concentration (MIC) are given in
µg/mL.
Organism MIC (µg/mL) Comment
Aspergillus fumigatus ATCC 204305
Aspergillus niger
7/10*
100*
Environmental pathogen, mycosis
Fruit pathogen (Black mole)
Botrytis cinerea CKJ1731 5 Grape vine/fruit rot
Candida albicans 3.5 Biofilm forming pathogen
Collectotrichum musae 12.5* Banana Pathogen
Cylindrocarpon liriodendri STEU 6170 3 Grape vine Black foot
Fusarum moliniforme
Fusarium solani STEU 6188
Fusarium oxysporum ATCC 10913
12.5*
9/12.5*
10
Rice and corn pathogen
Potato tuber rot, mycosis
Green leaf wilt
Fusarium verticilliodes CKJ1730 12 Corn blight, rot
Geothrichum citri-aurantii >50 Citrus pathogen, sour rot
Mucor piriformis 100* Mucor rot of fruits
Phaeoacremonium aleophilum <10 Young Grape vine decline
Penicillium expansum CKJ1733 5 Peach rot isolate
Penicillium expansum S 25 Apple rot, sensitive strain
Penicillium expansum R 100 Apple rot, resistant strain
Penicillium digitatum CKJ1734 4 Citrus rot isolate
Penicilium italicum S 12.5 Citrus rot, sensitive strain
Penicilium italicum R 12.5 Citrus rot, resistant strain
Penicillium glabrum CKJ1732 10 Wood isolate, strawberry pathogen
Phomopsis viticola <10 Grape vine leaf spot
Rhizopus stolonifer 100* Black bread mould
Talaromyces ramulosus CKJ1735 4 Peach rot isolate
Talaromyces mineoluteus CKJ1736 3 Peach rot isolate
Trichoderma atroviride 11 Wood isolate, bio-control agent
Natural Antimicrobial peptides in Agriculture