NATURAL ANTIMICROBIAL PEPTIDES AS GREEN MICROBICIDES … · Antimicrobial peptides (AMPs) are natural bio-control agents, which are part of the first line of defence of living organisms

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 AS GREEN MICROBICIDES 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

mailto:[email protected]


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.

mailto:[email protected]

http://www.sagreenfund.org.za/research


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


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


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


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.




Permanent staff member



Dr M Lutz Collaborator Collaboration on polymers and membranes

used for solid phase activity study Stellenbosch University


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


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


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


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


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.


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.


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.


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).


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).


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,


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


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


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


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.


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


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.


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.


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).


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


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.

http://www.toku-e.com/Tyrothricin-P732.aspx)

http://www.toku-e.com/Tyrothricin-P732.aspx)

Val-Gramicidin A 6.1

Val-Gramicidin B 0.6

Val-Gramicidin C 0.9


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


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


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


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


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


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)


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.


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).


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



plastic ware

Increase in

hydrophobicity,

antibacterial

Polystyrene

Synthetic

polymer

Various industrial &


plastic ware, glass

substitute

Character change

unknown

Poly(methyl

methacrylate

(PMM)

Synthetic

polymer

Various industrial &


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


industrial applications

Increase in

hydrophobicity,

antibacterial

Cellulose

acetate (CA)

Semi-

synthetic

polymer

Sterile filtration, various


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


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.


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)


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)


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.


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)


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.


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.


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.


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.


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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


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.


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


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.


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


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NATURAL ANTIMICROBIAL PEPTIDES AS GREEN MICROBICIDES … · Antimicrobial peptides (AMPs) are natural bio-control agents, which are part of the first line of defence of living organisms