1
SUSTAINABLE CONTROL OF PRE- AND
POSTHARVEST DISEASES OF CITRUS FRUIT
IN SOUTH AFRICA
Client: IDC APCF RESEARCH GRANT
FINAL REPORT
June 2018
Contact Persons:
Professor Mark D. Laing Office: 033 260 5526
Email: [email protected] Dr Iona H. Basdew
Office: 033 260 6475 Email: [email protected]
University of KwaZulu-Natal
Private Bag X01 Scottsville 3209
Pietermaritzburg
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Acknowledgements
The investigators would like to thank the following persons / organisations for the
significant contributions that they made to the study:
1. The Industrial Development Corporation (IDC) for their support during the study,
in particular Mr Kenneth Chauke and Mr Seatla Nkosi.
2. Mr Ken Woodburn of Stonelea Farms (Ixopo, KwaZulu-Natal) for allowing the
research team access to fruit trees and fresh fruit that were used during the
experiments.
3. The UKZN research team that comprised of research assistants, James Mathews,
Cleophas Chinheya, Gilmore Pambuka and Sanele Kubheka, and field assistants,
Thandazani Dlamini and Vincent Dlamini.
4. Dr Samson Tesfay (Horticulture Department, UKZN) for his advice on the plant
physiology aspects of the study.
5. The UKZN Finance and Administrative departments for their assistance over the
duration of the study.
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Executive Summary
a. Introduction
South Africa is currently 2nd in the world for citrus exports, after Spain.
Annual revenue - R9-10billion per annum.
80% is exported to the EU countries.
Current post-harvest losses in export revenue p.a. – approx. 45% = R4 – 4.5 billion
Losses to citrus black spot (Guignardia citricarpa)
o In November 2017 - the Citrus Growers Association banned all 2017 citrus
exports to the EU, except from the Western Cape and Northern Cape
(https://www.businesslive.co.za).
Primary postharvest losses: infection of citrus fruit by fungi / moulds:
o Penicillium digitatum (green mould)
o Penicillium italicum (blue mould)
o Geotrichum candidum (sour rot).
Current control of post-harvest Pencillium species: the fungicide imazalil
o Problem: difficult to get the right dose; residues, resistant strains in SA
Current control of post-harvest Geotrichum species: None
o the fungicide guazatine was banned in 2016 by the OECD countries.
Hypotheses Behind the Control Strategy:
Citrus fruits have an immune system, and can defend themselves from infection by
fungi.
A rapid hot water treatment (rHWT) (correct temperature x time) induces an
immune reaction in the fruit, releasing natural fungicides that kill most pre- and
post-harvest fungi.
Silicic acid inside plants (including fruit) mediates a faster, stronger immune reaction.
Silicic acid inside plants mediates enhanced resistance to abiotic stress such as
chilling injury and aging.
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b. Overall Objective
To develop alternative control strategies, integrated into a combined technology
Target CBS and the postharvest moulds of citrus
Apply control measures on farms and in packhouses, large and small
Use safe technologies (GRAS) , which opens up organic markets too
The integrated technology has to outperform existing technologies for cost and
efficacy, if farmers are to adopt it.
c. Specific Objectives
Test the effects of pre-harvest silicon fertilization of citrus on disease resistance and
cold tolerance.
Measure silicon uptake into citrus fruit.
Test the effects of pre-harvest silicon fertilization for protection against chilling
injury (esp. lemon and grapefruit).
Optimize a rapid hot water treatment (rHWT), postharvest, to eliminate prior
infections by fungi.
Optimize a citrus yeast (B13) as a biological control agent to prevent subsequent
postharvest infections
Evaluate the performance of the integrated technology: silicon fertilization + HWT +
Yeast versus the current industry standard, imazalil.
Determine the biochemical effects of the hot water treatment and yeast applications
Determine whether rHWT can reduce the development of citrus black spot (CBS)
from latent infections (the fungus initially infects the skin but leaves no symptoms).
d. Materials and Methods
Silicon Fertilization
Citrus varieties: Navel, Valencia and Eureka lemon
Silicon preparations: granular and liquid silicon fertilizers
Controls: water and potassium sulphate
Treatment period: 11 months
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Assessments:
o levels of silicon measured in citrus peels using Electron Dispersive Xray
analysis (SEM-EDX)
o chilling injury measured in Eureka lemon treated as for cold sterilization
versus fruit fly (0.5°C x 2-6 weeks in a cold room).
Control of Pathogens
Fruit picked from trees + or – silicon fertilization, washed, injured and inoculated
with green mould spores, then treated:
rHWT treatment: 60°C, 62°C, 64°C x 10, 15, 20 seconds
Control treatment: 20°C x 30 s
Biocontrol: Yeast B13
Assessments:
o Fruit assessed for disease development
o Fruit peels were quantitatively assessed for levels of plant protection
compounds associated with disease resistance.
Citrus black spot trials: different fruit used, from an unmanaged farm with high
levels of CBS. Field-infected Navel and lemon fruit picked, prior spots labelled, fruit
treated with HWT + B13. Assessment – number of new spots over time.
e. Results
Citrus varieties: all three citrus varieties reacted similarly to the treatments
Silicon fertilization: silicon was taken up into the fruit, from both liquid and granular
silicon sources
rHWT treatments + B13 – reduced levels of green mould in all three citrus fruit by
95-100%, whereas Control fruit had 98-100% infection levels.
Integrated Control: Silicon+rHWT+Yeast provided the best control.
Silicon + rHWT + Yeast: increased levels of natural plant protection compounds
(antioxidant proteins, phenolics and flavonoids).
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The mode of action of the integrated treatments:
o Silicic acid in citrus primes the fruit to accumulate more enzymes and
compounds for resistance, but does not trigger any reaction by itself.
o rHWT induce a disease resistance reaction in the fruit via heat-shock
proteins, which trigger off a cascade of defense reactions.
o The chemicals we measured are components of the disease resistance
cascade.
o Silicon + rHWT produce a faster, stronger resistance reaction than either
treatment alone.
o The B13 yeast coating then acts as a preventative barrier versus future
infections, stopping the spores of pathogens from germinating in wounds.
Lemons with pre-harvest silicon treatments were tolerant of chilling for up to 6
weeks at ±0.5°C.
Lemons with pre-harvest silicon treatments were also slower to dehydrate, retaining
their firmness and colour longer than control fruit.
Silicon was found in the skin of the fruit using SEM-EDX.
Citrus black spot control: rHWT of 68°C x 20 seconds stopped the development of
CBS spots in both lemon and orange between 90-98%, in highly infect fruit (worst
case scenario).
f. Research Conclusions
rHWT (640C x 15 seconds) + Yeast B13 control postharvest diseases, e.g., P.
digitatum, on lemon, and Navel and Valencia orange fruit. The integrated treatment
matched the level of disease control by the fungicide Imazalil.
The optimal rHWT combination of Temp x time varies slightly for each type of citrus.
Yeast B13 prevented future P. digitatum infections. It is not curative and needs to be
preceded by a hot water treatment to kill latent and prior infections.
Pre-harvest silicon treatments enhanced disease resistance and the rHWT+B13
active treatment versus postharvest diseases.
Pre-harvest silicon treatments conferred resistance to chilling injury in lemon fruit.
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On a global scale, the combination of silicon fertilization + rHWT treatment + yeast
biocontrol is a technology package that could be applied to all fruit and vegetable
crops to manage most postharvest diseases.
Technology Transfer Challenge – how to deliver this technology package to fresh
produce, flower and fruit farmers?
Prototype Packhouse Units: we have built a small scale rHWT + B13 unit, and a
medium scale unit. These both need to be optimized. We have designed a large scale
unit that can be retrofitted into existing mega-packhouses. This needs to be built and
optimized in real packhouses.
g. Future of the Project: Proposed Next Steps
Phase 2: Citrus Industry
Build the treatment plants, and transfer this technology into the citrus industry.
Small, medium and large scale packing operations all need the technology:
o Optimize sterilization washing step using anolyte water to replace HTH.
o Insert accurate, fast HWT step to replace slow, costly water bath/ fungicide
step.
o Dry the fruit, then coat with Yeast B13 before waxing and packing.
Commercialization pathway: various stakeholders ranging from growers,
packhouses, the CGA, commercialization partners and engineers.
Outcomes of Phase 2
o Enhance profitability of the export component
o Reduced risks for citrus farmers re pesticide use
o Reduced risks of fruit being rejected for pesticide residues or excessive
disease levels.
o Enhanced shelf life for the fruit
o Reduced costs of postharvest processing
o Organic status for the fruit at the processing stage
o Enhanced status of South African fruit, attracting a premium price
o Reduced risk of trade barriers by Spain such as the CBS issue
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o The creation of a new industry – the supply of equipment to implement
rHWT technology at the farm and packhouse
Phase 3: Postharvest Disease Management: Fresh Produce
Pasteurization revolutionized the shelflife of most liquid food products: milk, cream
beer, wine, fruit juice, medicines.
Think of this technology, rHWT, as the equivalent of pasteurization to treat all fresh
produce: Fruit, Vegetables, Flowers, Nuts, Seeds, Tubers, Bulbs, even sugar cane
stalks.
Because - the same hypothesis exists – that all plant organs can defend themselves,
and rHWT will induce a resistance reaction to cure prior infections; and selected,
safe yeasts can be used to stop future infections; and silicon fertilization can
enhance resistance to biotic and abiotic stress in most plant products. The resistance
reaction works against all normal pathogens.
We have initiated this research already:
o Tomato, avocado, cabbage and bean seed, groundnut
Multi-disciplinary approach:
o plant pathologists, entomologists, agricultural engineers and horticultural
scientists,
o industry partners who will take the technology to the greater audience.
Commercialization pathway: various stakeholders ranging from growers,
packhouses, the CGA, commercialization partners and engineers.
Outcomes of Phase 3:
o Enhance profitability of fresh produce farmers
o Reduced risks for farmers and consumers re pesticide use
o Reduced risks of food crops being rejected for pesticide residues or excessive
disease levels.
o Enhanced shelf life for the fresh produce (especially important for export
crops)
o Reduced costs of postharvest processing
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o Organic status for the fruit at the processing stage
o Enhanced status of South African fresh produce, attracting a premium price
o The creation of a new industry – the supply of equipment to implement
rHWT technology at fresh produce farms and packhouses.
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Table of Contents
Acknowledgements ................................................................................................................... 1
Executive Summary ................................................................................................................... 3
1. Introduction ......................................................................................................................... 12
2. Terms of Reference ............................................................................................................. 13
3. Report on Milestones .......................................................................................................... 14
3.1 Milestone 1: Development of improved postharvest treatment regimens for citrus in
packhouses (and in storage) in order to manage postharvest disease onset ..................... 14
a. Navel oranges ............................................................................................................... 14
b. Valencia oranges ........................................................................................................... 15
c. Research Conclusions for combined experiments ........................................................ 15
3.2 Milestone 2: Development of a disease management system for latent black spot
infections in orange and lemon ........................................................................................... 20
a. Navel oranges ............................................................................................................... 20
b. Eureka lemons .............................................................................................................. 21
c. Research Conclusions for CBS Control .......................................................................... 21
3.3 Milestone 3: Laboratory studies to aid the understanding of the protective mode of
action of silicon in leaves and fruit of citrus ........................................................................ 23
a. Antioxidants, phenolics, flavonoids .............................................................................. 24
b. Research Conclusions for silicon pretreatment and enhanced plant immunity .......... 28
3.4 Milestone 4: Development of improved preharvest treatment regimens for citrus
trees in order to manage preharvest disease onset ............................................................ 29
a. Silicon fertilisation and its effects on Navel, Valencia and Eureka ............................... 30
b. Silicon and chilling injury on Eureka lemon .................................................................. 33
c. General observations .................................................................................................... 34
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d. Research Conclusions for integrated treatments and chilling injury ........................... 35
3.5 MILESTONE 5: Publication of Research Findings ........................................................... 36
a. Journal Articles in Preparation ..................................................................................... 36
3.6 MILESTONE 6: University Administration ...................................................................... 37
4. Future of the Project ........................................................................................................... 37
APPENDIX ................................................................................................................................ 41
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1. Introduction
South Africa competes amongst the top countries in terms of world citrus export markets.
Losses in export revenue of more than 45% are incurred each year because of post-harvest
infection of citrus fruit (80-90% losses) intended for the European Union (EU) and Asian
markets (Montesinos-Herrero et al., 2009; Lesar, 2013; Christie, 2016). The greatest loss of
citrus fruit is a result of decay caused by the postharvest fungal pathogens Penicillium
digitatum (green mould), P. italicum (blue mould) and Geotrichum candidum (sour rot)
(Erasmus, 2014).
Postharvest damage through infection by these three pathogens is usually controlled using
fungicides, such as imazalil and guazatine (Erasmus, 2014). However, difficulties with
accurate application of the fungicides under less-than ideal conditions, resistance problems,
costs, and objections to fungicide residues by international markets, have all combined to
limit the viability of using postharvest fungicides as the sole control strategy. Resistance to
commonly used fungicides by key post-harvest pathogens is widespread and growing. There
are already strains of Penicillium species with resistance to all the currently registered
postharvest fungicides (Erasmus etal., 2015). Effective and reliable control of these
postharvest citrus diseases is paramount for this industry, so novel control strategies are
crucial.
Furthermore, export losses incurred through citrus black spot (Phyllosticta citricarpa) is a
topic frequently publicised in popular media. The EU has enforced exceptionally strict export
measures (5 positives and all exports will be banned), ostensibly because of the appearance
of this particular disease, and probably as a trade barrier, given that the pathogen was
historically known not to establish in the EU given their environmental conditions. However,
recent research conducted by Guarnaccia et al. (2017), signifies a first report of P. citricarpa
in Portugal and Italy (countries that are a part of the EU). This study however, does present
several anomalies, particularly around the mode of entry of the pathogen into the surveyed
orchards, and whether or not the isolated pathogen actually caused disease or lay latent in
orchard leaf litter. The study is currently under extensive review by the European Food
Safety Authority (EFSA) and by the National Plant Protection Organisation (NPPO), in order
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to confirm the findings presented by Guarnaccia et al. (2017) (EFSA PLH Panel, 2018). The
consequences of this study on South African export embargos has not be ascertained to
date. Speculatively, post-evaluations by EFSA may dictate new rules for South African citrus
exports to the EU, particularly with approximately 41% of the country’s citrus export
intended for European markets (ITC Trademap, 2017). Hence, the effective management of
CBS has received a resurrection and remains of paramount importance.
2. Terms of Reference
We propose the development of a control strategy aimed at the major postharvest fungal
diseases of citrus fruit during both the on-farm and first-tier facets of fruit processing, using
environmentally and consumer-friendly control measures, which are also cost-effective.
Prior research on control of pre- and postharvest diseases of citrus fruit suggests that the
application of a potassium silicate fertiliser in the orchard prior to harvest for enhanced
disease resistance, combined with novel treatments in the packing house, may provide a
viable alternative to fungicides. The novel integrated system involves the treatment of fruit
picked from treated trees with a rapid hot water treatment (HWT) (at temperatures in the
region of 50-60°C X 10-30 seconds), followed by application of a yeast biocontrol agent
(originally isolated from an orange).
It was also considered worthwhile to test the optimum HWTs for their ability to kill the
fungus causing citrus black spot (CBS) whilst it is in the latent state, before a black spot
lesion develops. This is the most dangerous situation for citrus exporters because they
cannot visually see these latent infections, which then develop in the 6 weeks that it takes
for fruit to travel to Europe in a container.
An unexpected bonus was the discovery that the application of soluble silicon fertiliser
pre-harvest improves the ability of citrus fruit to withstand chilling injury (Basdew and
Laing, unpublished data). This is of particular significance where fruit is exported to
markets where cold sterilisation (21 days at -0.5°C) is a requirement, in order to kill fruit fly
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larvae. The improvement of resistance to chilling injury is of particular importance to lemon
and grapefruit farmers because these citrus types are very sensitive to chilling injury.
Interestingly, heat treatments have also been recognised as a method for inducing
resistance to chilling injury. We may find synergy or additive effects when we combine in-
field fertilization with potassium silicate, and follow this with postharvest HWT of the citrus
fruit.
3. Report on Milestones
3.1 Milestone 1: Development of improved postharvest treatment regimens for
citrus in packhouses (and in storage) in order to manage postharvest disease
onset
The primary objective of Milestone 1 was to determine whether the application of rapid
HWTs plus the application of a topical biological control agent is able to manage disease
onset and severity in citrus fruit, postharvest. Fruit of the citrus varieties, Navel (c.v. Navel)
and Valencia (c.v. Midnight), were inoculated with spores of the fungus, Penicillium
digitatum (c.a. citrus green mould), then subjected to HWTs (HWT) (20°C, 58°C, 60°C, 62°C,
64°C, 66°C, 68°C x 10, 15, 20 seconds), followed by a post-HWT using a biocontrol coating
with a yeast labelled B13.
Note: Research was previously conducted on the effects of HWT on green mould of lemons
by Abraha (2008). Key findings included a delay in disease development up to 10 days post-
inoculation.
a. Navel oranges
In experiments testing HWT + B13 combinations on Navel oranges, reductions in disease
development ranging from 95-98% was observed at temperatures of 60°C and 64°C, when
used with the 15sec and 20sec HWT duration (Table 1, Figure 1). This was manifested by a
reduced numbers of diseased fruit per treatment, 2 weeks post-treatment. There was also
little variation in treatment efficacy based on the duration of treatments, i.e., treatment of
fruit for 10, 15 or 20 seconds did not differ significantly between treatments.
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This suggests that temperature is the principal determining factor for successful
treatments, and that the heat shock event takes place very rapidly, at the correct
temperature. This heat shock reaction appears to be followed by a host resistance reaction,
which then eliminates any pathogen present in the fruit. The resistance reaction has a
limited life of about 3 days. The Yeast B13 treatment follow HWT, and to prevent future
infections during postharvest processes between the packhouse and the consumer. Most
importantly, the experimental fruit treated with HWT consistently showed no physical
evidence of heat damage, throughout the duration of the experiment (2 weeks), and four
weeks later (during which time fruit were kept at ambient temperature in the postharvest
experimental laboratory; a total of six weeks for entire duration of experiment).
b. Valencia oranges
Experiments on Valencia oranges (c.v. Midnight) were also successfully conducted. Midnight
oranges appeared highly resistant to infection, as manifested by significantly higher
percentage of uninfected fruit, even three weeks post treatments (Table 1, Figure 2).
Optimal HWTs were in the range 62°C-64°C for 10-20s with a reduction in disease
development of between 95-98%, which was consistent with those results derived from the
Navel trials. As with Navels, Valencia oranges showed no physical evidence of heating
damage due to the rapid HWT treatments.
c. Research Conclusions for combined experiments
All results were consistent over three blocks of trials conducted, early, middle and
late in the season, for both Navel and Valencia.
There were no within-season differences noted throughout the trial, i.e., fruit were
picked during early-season, mid-season and late-season for each trial that was
conducted, hence three sets of experiments were conducted per fruiting season, per
citrus variety. The use of B13 in addition to HWT provided superior control versus
HWT alone.
In general, Valencia fruit were able to tolerate HWTs at higher temperatures than
Navel fruit can tolerate without damage.
However, fruit treated at 64-66°C for both varieties of citrus showed no signs or
symptoms of disease even after 4 weeks, post-treatment, whereas those fruit
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treated at temperature ranging from 58-60°C and 68°C developed disease
approximately 2 weeks post-treatment.
In the experimentation conducted, the specific mode of action of B13 that has been
exploited is its ability to colonise the fruit surface and thereby outcompete any
secondary microbes from colonizing the primary plant material. It is this
preventative action, rather than a curative one, that distinguishes the spectrum of
activity of B13 versus a fungicide. This is a living organism that will actively colonise
any injury or intrusion on the fruit surface, thereby preventing the entry and
accumulation of secondary organisms.
The ability to control post-harvest diseases by HWT+B13 can rival treatment of fruit with
Imazalil alone. Levels of control provided by the best HWT+B13 treatments, and the use of
Imazalil alone, were not statistically different.
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Table 1. Levels of green mould that developed on inoculated Valencia fruit that were subjected to HWT, and treated, or not treated, with B13.
Hot water x Time % Navel oranges infected % Valencia oranges infected
(°C x s) - B13 + B13 - B13 + B13
20°C 5s 18 12 28 23
20°C 10s 28 15 33 19
20°C 15s 8 5 30 8
20°C 20s 23 8 25 10
58°C 5s 15 5 28 9
58°C 10s 8 8 23 8
58°C 15s 13 10 13 9
58°C 20s 10 8 13 13
60°C 5s 20 12 10 9
60°C 10s 10 7 25 7
60°C 15s 5 8 5 9
60°C 20s 10 15 3 11
62°C 5s 23 5* 18 6*
62°C 10s 20 8* 5 8*
62°C 15s 18 10* 13 8*
62°C 20s 13 8* 13 8*
64°C 5s 40 5* 3 8*
64°C 10s 25 3* 8 5*
64°C 15s 18 3* 3 6*
64°C 20s 30 3* 5 5*
66°C 5s 28 8 13 12
66°C 10s 23 10 5 15
66°C 15s 15 27 5 23
66°C 20s 13 52 8 33
68°C 5s 25 58 28 37
68°C 10s 13 37 13 25
68°C 15s 5 50 8 36
68°C 20s 8 65 13 43
Cont1* inoculated 88 12 88 12
Cont2** uninoculated 0 8 0 8
Cont3***Imazalil 0 0 0 0
*Cont1=inoculated, untreated fruit; **Cont2=uninoculated, untreated fruit; ***Cont3=fruit treated with Imazalil.
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-2
0
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6
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Ave
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ruit
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Temperature ( C) * Time (sec)
Infected
Uninfected
Figure 1. Mean number of infected versus uninfected Navel oranges; fruit were subjected to HWTs and then treated with B13 (Cont1=inoculated, untreated fruit;
Cont2=uninoculated, untreated fruit; Cont3=fruit treated with Imazalil).
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-2.00
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
Aver
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Infected
Uninfected
Figure 2. Infected versus uninfected Valencia oranges after HWT + B13 (Cont1=inoculated, untreated fruit; Con2=uninoculated, untreated fruit; Cont3=fruit treated with Imazalil); Data is an average of three blocks of tria
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3.2 Milestone 2: Development of a disease management system for latent black
spot infections in orange and lemon
The citrus black spot (CBS) trial was conducted in a pilot study to prove the concept that the
HWT can kill the CBS fungus present in latent infections of citrus fruit. The research protocol
involved marking already present disease spots on Navel oranges and Eureka lemons (Figure
3), and then counting any new spots that developed over time. Any newly developing
lesions that were visible at the time that the fruit were marked, were also marked and
counted at the start of the trial This method did not use a controlled inoculation of
individual sample fruit because G. citricarpa infects the citrus fruit at fruit-set (and even
during flowering), and infection remains dormant until fruit are mature. As a result, the
exact levels of latent infection of fruit cannot be accurately predicted from prior
inoculations. Our experimental protocol represents a worst-case scenario for a citrus
packhouse, by using fruit that already had visible lesions, and therefore had a very high
likelihood of latent infections.
Figure 3: Citrus black spot lesions on Navel orange (left) and Eureka lemon (right).
a. Navel oranges
A general trend across all sets of trials for Navel oranges was that treatment of fruit at
combinations of between 56°C x 15 seconds to 58°C x 10 seconds, caused the greatest
reduction in latent infection of black spot (Table 2). Temperature x Time treatment
combinations at temperatures greater than 62°C caused an increase in infection, possibly as
a result of damage to the fruit’s resistance system. Where this resistance was not
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suppressed by high temperatures, the effects of fruit resistance induction could be seen up
to 9 days post-treatment, with a reduction in the development of new CBS lesions (Table 2).
Furthermore, treatment of fruit early and middle in the season, at 56°C x 10 seconds to 60°C
x 15 seconds, showed a reduction in disease development, disease control between 98-
100% (Appendix: Figures 1, 2, 3). The times of the season at which fruit were picked and
treated also played a significant role in disease management. Picking fruit earlier in the
season and treating these fruit earlier, caused a significant reduction in development of
latent infection, whereas the batch of fruit picked late in the season showed a lesser
reduction in lesion development over the 9d screening period.
b. Eureka lemons
This trial differs from the trial carried out on Navel oranges in terms of the method of
disease rating. Infection was severe on the lemons throughout the season, hence individual
spot counts were not possible. As a result, infection was rated as a cumulative percentage of
the fruit covered by lesions from Day 6, Day 10 and Day 14, post treatment. A general trend
across all three sets of trials was that the treatment of fruit at temperatures between 66°C x
20 seconds to 68°C x 20 seconds caused the greatest reduction in latent infection (Table 3).
Temperature x Time treatment combinations at less than 66°C did not appear to suppress
lesion development. The times of the season at which fruit were picked and treated played
a significant role in disease management. The lemon results were similar to those of the
Navel oranges (Appendix: Figures 4, 5, 6). Lemons that were picked early and mid-season,
manifested reduced disease symptoms across the entire sampling period (14 days) as
demonstrated by a reduction in black spot lesion development relative to the untreated
controls. However, fruit that were picked later in the season showed reductions in lesions
development ranging from 22-78%.
c. Research Conclusions for CBS Control
Initial results show that intensive bursts of HWT (e.g., 680C x 20 seconds) were able
to reduce latent CBS development in both lemon and orange, but did not provide
100% control of such latent infection, in a highly infected batch of fruit.
Therefore, an HWT treatment of lightly infected fruit could control close to 100% of
the latent infections of CBS.
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This HWT treatment ties in with the control of other latent infections such as blue
mould, so the non-chemical control of a wide range of postharvest diseases of citrus
using HWT should be feasible.
Table 2*. Mean black spot counts on Navel oranges on Day 0 (before treatment) in
comparison to a cumulative black spot counts from Day 5 to Day 9 (post-treatment), across
the season.
Temp x Time Mean spot counts Day 0 Mean spot counts from Day 5 to Day 9
20°C5s 10.111 abc 14.889 hi
20°C10s 8 abc 13.089 ghi
20°C15s 10 abc 11.733 efgh
52°C5s 5.622 a 9.511 defg
52°C10s 8.422 abc 6.222 abcd
52°C15s 7.067 abc 4.867 abc
54°C5s 7.222 abc 4.022 abc
54°C10s 10.778 c 4.489 abc
54°C15s 7.711 abc 5.444 abcd
56°C5s 5.467 a 3.444 ab
56°C10s 9.067 abc 5.089 abcd
56°C15s 7.622 abc 3.756 ab
58°C5s 9.889 abc 3.956 abc
58°C10s 7.733 abc 2.911 a
58°C15s 6.511 abc 4.022 abc
60°C5s 5.778 ab 4.867 abc
60°C10s 7.778 abc 5.022 abcd
60°C15s 7.511 abc 5.133 abcd
62°C5s 9.489 abc 16.267 i
62°C10s 9.556 abc 8.533 cdef
62°C15s 10.622 c 12.222 fghi
64°C5s 9.444 abc 7.978 bcde
64°C10s 10.111 abc 13.889 hi
64°C15s 10.556 bc 7.316 abcd
66°C5s 10.811 c 8.067 bcde
66°C15s 7.8 abc 12.489 fghi
66°C10s 8 a 6.911 abcd
68°C5s 7.667 abc 6.844 abcd
68°C10s 8.244 abc 6.956 abcd
68°C15s 9.911 abc 6.578 abcd
Control 6.27 ab 7.5 abc
*Note: Figures 1, 2 and 3 in the Appendix are visual representations of the data contained herein.
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Table 3*. Percentage infection of Eureka lemons by citrus black spot on Day 0 (before
treatment) in comparison to cumulative black spot development from Day 6 to Day 14
(post-treatment), across the season.
Temp x Time Day0_ %infection Cumulative lesion development day
6 to Day 14, post treatment
20°C5s 50.33 abcd 45.67 l
20°C10s 51.33 abcd 44.33 kl
20°C15s 59.67 bcdef 32.67 ghij
20°C20s 44.33 a 42.67 jkl
58°C5s 48.00 abc 34.00 hijk
58°C10s 52.67 abcd 22.00 bcdefgh
58°C15s 43.67 a 35.33 ijkl
58°C20s 51.33 abcd 30.00 efghi
60°C5s 55.00 abcd 26.33 defghi
60°C10s 56.33 abcde 30.33 efghi
60°C15s 52.33 abcd 31.00 fghi
60°C20s 49.33 abcd 26.33 defghi
62°C5s 61.67 cdef 26.00 defghi
62°C10s 64.00 def 24.67 cdefghi
62°C15s 59.67 bcdef 27.00 defghi
62°C20s 60.33 bcdef 13.67 abc
64°C5s 60.00 bcdef 21.33 bcdefg
64°C10s 56.67 abcde 21.33 bcdefg
64°C15s 51.67 abcd 23.00 bcdefgh
64°C20s 45.33 ab 19.33 bcdef
66°C5s 71.33 f 18.67 abcde
66°C10s 63.00 cdef 16.00 abcd
66°C15s 60.33 cdef 13.33 abc
66°C20s 60.00 bcdef 12.33 ab
68°C5s 70.67 ef 13.00 abc
68°C10s 50.33 abcd 19.33 bcdef
68°C15s 54.67 abcd 16.00 abcd
68°C20s 48.00 abc 7.00 a
control1 49.33 abc 40.00 hij
*Note: Figures 4, 5 and 6 in the Appendix are visual representations of the data contained herein.
3.3 Milestone 3: Laboratory studies to aid the understanding of the protective
mode of action of silicon in leaves and fruit of citrus
24
The objective of this study was to determine whether the application of preharvest
treatments to citrus trees is able to influence antioxidant production in citrus fruit,
postharvest, and also to investigate whether or not silicon that is taken up by the roots of
the tree can be deposited into the fruit itself. The preharvest protocol involved treatment of
Navel citrus trees with silicon fertiliser for a period of 18 months. Four preharvest
treatments were applied: potassium silicate in a liquid form (KSil-Liq), potassium silicate in a
granular form (KSil-Gran), Water (control 1), Potassium Sulphate (control 2). During the
postharvest experimentation, fruit collected from these treated trees were inoculated with
fungal spores followed by HWTs at 60°C, 62°C, 64°C x 15 or 20 secs. Fruit were then dipped
in a coating treatment using a yeast biological control agent, B13 (Candida fermentati). Fruit
were left for 24 hours, before extraction and analysis of fruit peel exudates was carried out.
Peel exudates were assayed for the presence of flavonoids, antioxidants and phenolics.
Silicon uptake was assessed in the flavedo of the fruit using scanning electron microscopy
(energy dispersive x-ray microscopy).
a. Antioxidants, phenolics, flavonoids
Our studies showed a marked increase in the levels of plant protective compounds in fruit
that received silicon pretreatments. However, there were certain anomalies that were also
noted. Particularly, with reference to the significantly higher levels of antioxidants in the
control experiments, where the fruit were either inoculated and treated with a fungicide, or
only inoculated (Figure 4). We have yet to determine whether this trend was a result of an
outlier or if it is indeed significant.
Levels of phenolics and flavonoids in fruit peels from all silicon pre-treated experiments
were significantly higher than the fruit peels of the control treatments (potassium sulphate
or water pretreatment) (Figures 5, 6). The amount of silicon deposited from tree to fruit was
significantly higher in silicon pre-treated trees, than fruit from control trees (Table 4). A
noteworthy observation however, was that although control trees did not receive silicon
pretreatments, the fruit did contain residual, micro-amounts of naturally acquired silicon.
25
Figure 4: Antioxidant levels in fruit peels. Fruit picked from pre-treated trees, and subjected to postharvest treatments; C1 = fruit inoculated +
Imazalil, C2 = fruit inoculated (no other treatment), C3 = fruit uninoculated and untreated.
26
Figure 5: Phenolic levels in fruit peels. Fruit picked from pre-treated trees, and subjected to postharvest treatments. C1 = fruit inoculated +
Imazalil, C2 = fruit inoculated (no other treatment), C3 = fruit uninoculated and untreated.
27
Figure 6: Flavonoid levels in fruit peels. Fruit picked from pre-treated trees, and subjected to postharvest treatments. C1 = fruit inoculated +
Imazalil, C2 = fruit inoculated (no other treatment), C3 = fruit uninoculated and untreated
28
Table 4: Major elemental composition of citrus fruit peels, esp. potassium silicate (Si)
b. Research Conclusions for silicon pretreatment and enhanced plant immunity
Firstly, we confirmed that silicon is taken up by citrus plants via their roots, and
transported to their fruit.
Secondly, silicon treatment of citrus trees, combined with HWT of citrus fruit,
enhanced levels of antioxidant proteins, phenolics and flavonoids in fruit, which are
indicators of a resistance response to fungal infection.
Pretreatment of citrus trees with silicon fertilizers primes the trees to accumulate
more enzymes and compounds for resistance, but does not trigger any reaction by
itself.
HWT induces / triggers the resistance reaction through the stimulation of heat-shock
proteins, which in turn, trigger off a cascade of defense reactions.
The chemicals we measures contribute to the resistance cascade.
Silicon plus HWT, together produce a faster, stronger resistance reaction than either
treatment alone.
Treatment Major elemental composition of fruit flavedo (%) – nothing NB
KSil-Liq Si C O P K Ca
Navel 3.12 52.66 48.92 0.03 0.25 0.08
Lemon 1.30 41.89 46.12 0.06 7.11 0.25
KSil-Gran
Navel 0.25 56.26 43.13 0.01 0.17 0.20
Lemon 0.76 48.72 49.75 0.02 6.43 0.41
PotS
Navel 0.07 58.12 41.37 0.03 0.22 0.14
Lemon 0.15 50.64 48.01 0.06 0.72 7.00
Control
Navel 0 51.07 35.84 0.07 1.06 2.05
Lemon 0.07 46.89 44.92 0.06 0.62 3.85
29
3.4 Milestone 4: Development of improved preharvest treatment regimens for
citrus trees in order to manage preharvest disease onset
The objective of this facet of the research was to determine whether preharvest treatments
of citrus trees using silicon fertilisers could deliver a protective effect on fruit against citrus
green mould caused by the fungus Penicillium digitatum (Figure 7), postharvest, and
secondly, whether they could confer/enhance tolerance to chilling injury in lemons.
Figure 7. Penicillium digitatum on oranges (a), P. digitatum spores under the light
microscope at 1000x magnification (b).
The preharvest protocol involved treatment of Navel, Valencia and Eureka citrus trees with
silicon fertilisers for a period of 18 months. Two different silicon preparations were used,
i.e., potassium silicate in a liquid form (KSil-Liq) and potassium silicate in a granular form
(KSil-Gran); control treatments included water and potassium sulphate. Fruit collected from
these treated trees were then subjected to HWT+B13 during the postharvest
experimentation (Figure 8). The fruit were inoculated with spores of P. digitatum (Figure 8),
then treated at 60°C, 62°C or 64°C x 15 or 20 secs. This was followed by fruit coatings using
the yeast biological control agent called B13 (Candida fermentati). Fruit were then assessed
for disease development, two weeks post-treatments.
a b
30
Figure 8: Postharvest fruit treatment protocol.
a. Silicon fertilisation and its effects on Navel, Valencia and Eureka
Fruit picked from trees that were treated with either KSil-Liq or KSil-Gran, preharvest,
showed superior resistance to infection by green mould, compared with the control
treatments, when subjected to hot water and yeast treatments, postharvest (Table 5).
Lemons in general, showed minimal susceptibility to green mould throughout the trials.
Overall results for all three citrus varieties show that silicon-based preharvest treatments,
combined with postharvest HWT + B13 treatments, conferred greater resistance to PD in
citrus fruit (Appendix: Figures 7, 8, 9, 10, 11,12) .
31
The levels of protection of fruit across all three varieties ranged from 95-100% protection.
These observations were further reinforced by the performance of the control treatments,
particularly Control 2 (inoculated), where high levels of disease incidence was noted in fruit
that received no postharvest treatments.
The mode of action is that silicon application pre-harvest, primes the plant for
enhanced defence against biotic stresses. The artificial inoculation of fruit with a foreign
body (fungal spore), coupled with the HWT, triggers the plant defences, which then
deliver a cascade of antifungal compounds, destroying the pathogen, whether latent or
actively infecting the fruit. The B13 yeast coating then acts a preventative barrier to
future infection, stopping the spores of pathogens from germinating in wounds.
32
Table 5: Percentage of fruit uninfected across all treatments (pre- and postharvest), across all varieties
Percentage of citrus fruit uninfected across all treatments (pre- and
postharvest), across all varieties
Fruit Uninfected (%)
NAVEL VALENCIA LEMON
Inoculated Treatments KSil-
Liq
KSil-
Gran
PotS Control KSil-
Liq
KSil-
Gran
PotS Control KSil-
Liq
KSil-
Gran
PotS Control
Plus B13 60°C x 20sec 97 100 98 95 100 98 88 98 100 100 100 100
62°C x 20sec 98 95 95 100 9598 98 93 100 100 100 100 98
64°C x 20sec 97 98 95 100 100 100 98 98 100 98 100 98
No B13 60°C x 20sec 95 100 98 100 95 100 88 100 98 98 100 100
62°C x 20sec 95 98 90 100 98 95 93 98 100 95 98 92
64°C x 20sec 100 95 92 98 100 98 95 95 98 98 100 98
Cont 1 – Inoc + Imazalil 100 100 100 92 100 100 100 100 100 100 100 100
Cont 2 - Inoc + untreated 13 100 8 98 70 50 30 20 60 40 0 20
Cont 3 - Uninoc +
untreated
93 95 98 0 100 100 100 100 90 100 90 90
33
b. Silicon and chilling injury on Eureka lemon
Fruit picked from lemon trees that received preharvest silicon treatments, either in liquid
form or granular form, showed superior tolerance to chilling injury (Figure 9, 10). Fruit
picked from control trees that received either potassium sulphate or plain water,
succumbed to chilling injury far quicker and far more severely, rendering them un-
marketable in a real-time export situation. Furthermore, silicon uptake was assessed in the
flavedo of the fruit using scanning electron microscopy (Table 6). This confirmed that silicon
is taken up by the plant and deposited into fruit, and it is postulated that this increased level
in silicon in the flavedo is able to confer chilling injury protection. Preharvest silicon
treatments also appeared to predispose the fruit to dehydration tolerance. Fruit from the
control groups appeared to shrink and brown faster than fruit subjected to preharvest
silicon treatments.
Figure 9: Rind injury assessment of Eureka lemons, over 6 weeks, after 2 week storage at +/-
0.5°C. Lemons were subjected to preharvest treatments prior to cold storage. Data is an
average of three blocks of trials x 2 seasons (2014 & 2015) = 6 pooled assessments.
34
Figure 10: Chilling injury on Lemon in Week 3 of storage. Note the shrinking and browning of the Control fruit.
Table 6. Elemental composition of fruit flavedo, with emphasis on silicon
Treatment Major elemental composition of fruit flavedo (%)
Si C O P K Ca
KSil-Liq 1.30 49.89 61.22 0.06 7.11 0.25
KSil-Gran 0.76 48.72 62.91 0.02 6.43 0.41
PotS 0.15 50.64 48.01 0.06 0.72 7.00
Control 0.07 49.89 48.92 0.06 0.62 3.85
c. General observations
It has been postulated that silicon uptake primes the plant by enhancing its resistance to
biotic and abiotic stresses (Deshmukh et al., 2017; Etesami and Jeong, 2018). Typical
mechanisms of protection include enhanced resistance through (1) deposition of silicon
35
beneath the cuticle, which acts as a physical barrier against either biotic or abiotic stresses
(“bullet proof”); (2) modulating host resistance systems by regulating the production of
protective compounds such as phenolics, antioxidants and phytoalexins. Furthermore, we
believe that the HWT acts synergistically with the silicon, by inducing the full resistance
reaction that the silicon has enhanced. This triggers a systemic acquired resistance reaction
within the fruit, an effect across the entire fruit. This process includes the production of
pathogenesis-related proteins, oxidative enzymes and phytoalexins, which act in concert to
kill any infectious agent.
In a nutshell, silicon-based pretreatments appear to physically prepare the plant to defend
against an attack by a pathogen, or then to tolerate an abiotic stress. By following Si-pre-
treatments with HWT, the maximum expression of host defense reactions are triggered,
resulting in optimal protection.
d. Research Conclusions for integrated treatments and chilling injury
HWTs at high temperature for a short durations, in combination with a yeast
biocontrol treatment, are able to combat postharvest diseases such as P. digitatum,
on both Navel and Valencia fruit. This combined treatment provided levels of
protection that matched those provided by the fungicide Imazalil.
The optimal HWT Time x Temperature combination will be slightly different for each
types of citrus. This suggests that all types of citrus need to be assessed to determine
the precise Time x Temperature combinations to ensure optimal control of
postharvest diseases.
Yeast B13 prevented P. digitatum infections, on a preventative basis. It is not
curative and needs to be preceded by a HWT to kill latent and prior infections.
Preharvest silicon treatments were shown to confer tolerance to chilling injury in
lemon fruit.
These developments have shown that a revision of current methods of postharvest
fruit treatment can save both time and resources, while still in keeping with “green”
technologies.
36
On a global scale, the combination of silicon fertilization + HWT + yeast biocontrol is
a technology package that could be applied to all fruit and vegetable crops to
manage most postharvest diseases.
What is needed is to convert the research outcomes in a functional technology
package that farmers can access.
We have initiated studies to create the machines to deliver these treatments
efficiently. We have developed prototypes of a small scale HWT + B13 unit, and a
medium scale unit. These both need to be optimized. There is also the need to
develop a large scale unit that can be retrofitted into existing mega-packhouses; this
needs to be designed, built and tested in the field.
3.5 MILESTONE 5: Publication of Research Findings
a. Journal Articles in Preparation
1. The effect of augmented pre- and postharvest treatments against Penicillium
digitatum in Navel oranges: Postharvest Biology and Technology
2. The effect of augmented pre- and postharvest treatments against Penicillium
italicum in Valencia oranges: Journal of Plant Diseases and Protection
3. The effect of augmented pre- and postharvest treatments against Penicillium
italicum in Eureka lemons: Biological Horticulture and Agriculture
4. The control of latent infections of citrus black spot using an integrated pre- and
postharvest treatment regimen: Horticultural Science
5. The enzymatic effect of silicon uptake in citrus and its ability to minimize disease
development in Navel oranges: Horticulture, Environment and Biotechnology
6. The enzymatic effect of silicon uptake in citrus and its ability to minimize disease
development in Valencia oranges: Hortscience
7. The enzymatic effect of silicon uptake in Eureka lemons and its ability to minimize
disease development and confer cold tolerance, postharvest: Horticulture Journal
37
3.6 MILESTONE 6: University Administration
Project administration via systems of operation at the University of KwaZulu-Natal have
been ongoing since the project inception. The different departments actively involved in the
daily running of the project include:
Principal Investigator’s Office
Postgraduate Office
UKZN Research Office
Contracts Office
Human Resources
Inqubate
College Finance Department
College Buying Office
College Higher Degrees Office
4. Future of the Project
The long term plan for the project is to implement the amended postharvest protocols into
the current practice applied by the citrus industry. Proposed revisions are primarily targeted
at reducing current fruit exposure times to HWTs from 5-15mins to 20sec, fruit coating post-
HWTs with a biological control agent (yeast B13) instead of the chemical fungicides
currently used (Imazalil), and also preharvest treatments that can be implemented in-field
(specifically silicon fertilization in-field) (Figure 11).
38
Figure 11: Proposed fruit treatment pathway, postharvest (proposed areas for amendments
are highlighted in green).
39
References
1. Abraha, A.O. (2008). Integrated use of yeast, Bacillus, hot water and potassium
silicate treatments for the control of postharvest green mould of citrus and litchis.
PhD thesis, University of KwaZulu-Natal.
2. Christie, C. (2016). Optimisation of postharvest drench application of fungicides on
citrus fruit. Thesis: Master of Science. Stellenbosch University.
3. Deshmukh, R.K., Ma, J.F., Belanger, R.R. (2017). Editorial: Role of Silicon in Plants.
Frontiers in Plant Science 8, pp. 1858, doi: 10.3389/fpls.2017.01858.
4. EFSA PLH Panel (EFSA Panel on Plant Health): Jeger, M., Bragard, C., Caffier, D.,
Candresse, T., Chatzivassiliou, E., Dehnen-Schmutz, K., Gilioli, G., Gr�egoire, J-C.,
Jaques Miret, J.A., MacLeod, A., Navajas Navarro, M., Niere, B., Parnell, S., Potting,
R., Rafoss, T., Rossi, V., Urek, G., Van Bruggen, A., Van Der Werf, W., West, J., Winter,
S., Baker, R., Fraaije, B., Vicent, A., Behring, C., Mosbach Schulz, O., Stancanelli, G.
(2018). Scientific Opinion on the evaluation of a paper by Guarnaccia et al. (2017) on
the first report of Phyllosticta citricarpa in Europe. EFSA Journal 16 (1):5114, 48 pp.
https://doi.org/10.2903/j.efsa. 2018.5114.
5. Erasmus, A. (2014). Optimisation of Imazalil application and green mould control in
South African packhouses. PhD thesis, Stellenbosch University.
6. Erasmus, A., Lennox, C., Korsten, L., Lesar, K., Fourie, P.H. (2015). Imazalil resistance
in Penicillium digitatum and P. italicum causing citrus postharvest green and blue
mould: Impact and options. Postharvest Biology and Technology 107, pp. 66-76.
https://doi.org/10.1016/j.postharvbio.2015.05.008.
7. Etessami, H., Jeong, B.R. (2018). Silicon (Si): Review and future prospects on the
actions mechanisms in alleviating biotic and abiotic stresses in plants. Ecotoxicology
and Environmental Safety 147, pp. 881-896, doi:
https://doi.org/10.1016/j.ecoenv.2017.09.063.
8. Guarnaccia, V., Groenewald, J.Z., Li, H., Glienke, C., Carstens, E., Hattingh, V., Fourie,
P.H., Crous, P.W. (2017). First report of Phyllosticta citricarpa and description of two
40
new species, P. paracapitalensis and P. paracitricarpa, from citrus in Europe. Studies
in Mycology 87, pp. 161-185.
9. ITC Trademap. (2017). South African citrus exports under the SADC-EU EPA.
https://www.tralac.org/discussions/article/12282-south-african-citrus-exports-
under-the-sadc-eu-epa.html
10. Lesar, K.H. (2013). Compendium of postharvest citrus diseases – illustrated. Citrus
Research International, Nelspruit, pp. 12.
11. Montesinos-Herreros, C., Angel del Rio, M., Pastor, C., Brunetti, O. Palou, L. (2009).
Evaluation of brief potassium sorbate dips to control postharvest Penicillium decay
on major citrus species and cultivars. Postharvest Biology and Technology 52(1), pp.
117–125.
41
APPENDIX
-2
0
2
4
6
8
10
12
14
16
18
Ave
rag
e s
po
t c
ou
nts
ove
r ti
me
(n
um
be
r o
f b
lac
k s
po
t le
sio
ns
pe
r fr
uit
)
Temperature x Time treatments
Day0_spot count Day5_spot count Day7_spot count Day9_spot count
Figure 1: Temperature x Time treatment of Navel oranges infected with citrus black spot; fruit were picked early in the growing season (mid May
2013); development of new lesions was tracked over 10 days(Duncan’s multiple range test where p≤0.05).
42
-2
0
2
4
6
8
10
12
14
16
18
20
Ave
rag
e s
po
t c
ou
nts
ove
r ti
me
(n
um
be
r o
f b
lac
k s
po
t le
sio
ns
pe
r fr
uit
)
Temperature x Time treatments
Day0_spot count Day5_spot count Day7_spot count Day9_spot count
Figure 2. Temperature x Time treatment of Navel oranges infected with citrus black spot; fruit were picked in the middle of the growing season
(early July 2013); development of new lesions was tracked over 10 days (Duncan’s multiple range test where p≤0.05).
43
-2
0
2
4
6
8
10
12
14
16
18
20
Avera
ge
sp
ot
co
un
ts o
ver
tim
e (
nu
mb
er
of
bla
ck s
po
t le
sio
ns
pe
r fr
uit
)
Temperature x Time treatmentsDay0_spot count Day5_spot count Day7_spot count Day9_spot count
Figure 3. Temperature x Time treatment of Navel oranges infected with citrus black spot; fruit were picked late in the growing season (mid-August
2013); development of new lesions was tracked over 10 days (Duncan’s multiple range test where p≤0.05).
44
-
10
20
30
40
50
60
70
20°C5s 20°C10s 20°C15s 20°C20s 58°C5s 58°C10s 58°C15s 58°C20s 60°C5s 60°C10s 60°C15s 60°C20s 62°C5s 62°C10s 62°C15s 62°C20s 64°C5s 64°C10s 64°C15s 64°C20s 66°C5s 66°C10s 66°C15s 66°C20s 68°C5s 68°C10s 68°C15s 68°C20s control1
% D
ise
as
e d
eve
lop
me
nt
ove
r t
ime
Temperature x Time treatments
Day0_%Infection Day6_%Infection Day10_%Infection Day14_%Infection
Figure 4. Temperature x Time treatment of Eureka lemons infected with citrus black spot; fruit were picked early in the growing season (mid May
2013); development of new lesions was tracked over 14 days (Duncan’s multiple range test where p≤0.05).
45
0
10
20
30
40
50
60
70
80
90
100
20°C5s 20°C10s 20°C15s 20°C20s 58°C5s 58°C10s 58°C15s 58°C20s 60°C5s 60°C10s 60°C15s 60°C20s 62°C5s 62°C10s 62°C15s 62°C20s 64°C5s 64°C10s 64°C15s 64°C20s 66°C5s 66°C10s 66°C15s 66°C20s 68°C5s 68°C10s 68°C15s 68°C20s control1
% D
ise
as
e d
eve
lop
me
nt o
ve
r t
ime
Temperature x Time treatments
Day0_%Infection Day6_%Infection Day10_%Infection Day14_%Infection
Figure 5. Temperature x Time treatment of Eureka lemons infected with citrus black spot; fruit were picked in the middle of the growing season
(early July 2013); development of new lesions was tracked over 14 days (Duncan’s multiple range test where p≤0.05).
46
0
10
20
30
40
50
60
70
80
90%
D
ise
as
e d
eve
lop
me
nt o
ve
r t
ime
Temperature x Time treatments
Day0_%Infection Day6_%Infection Day10_%Infection Day14_%Infection
Figure 6. Temperature x Time treatment of Eureka lemons infected with citrus black spot; fruit were picked late in the growing season (mid-August
2013); development of new lesions was tracked over 14 days.
47
Figure 7: Pretreatment of citrus trees with KSil-Liquid, followed by postharvest treatment of fruit with HWT and biological control agent,
Yeast B13; citrus cultivar: Navel.
48
Figure 8: Pretreatment of citrus trees with water only (in-field control), followed by postharvest treatment of fruit with HWT and biological
control agent, Yeast B13; citrus cultivar: Navel.
49
Figure 9: Pretreatment of citrus trees with KSil-Liquid, followed by postharvest treatment of fruit with HWT and biological control agent,
Yeast B13; citrus cultivar: Valencia.
50
Figure 10: Pretreatment of citrus trees with water only (in-field control), followed by postharvest treatment of fruit with HWT and biological
control agent, Yeast B13; citrus cultivar: Navel.
51
Figure 11: Pretreatment of citrus trees with KSil-Liquid, followed by postharvest treatment of fruit with HWT and biological control agent,
Yeast B13; citrus cultivar: Eureka.
52
Figure 12: Pretreatment of citrus trees with water only (in-field control), followed by postharvest treatment of fruit with HWT and biological
control agent, Yeast B13; citrus cultivar: Eureka.
