Identifying Volatile Emissions Associated with False Codling Moth Infested Citrus Fruit

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IDENTIFYING VOLATILE EMISSIONS ASSOCIATED WITH FALSE CODLING MOTH INFESTED CITRUS FRUIT

Rachel van der Walt

Submitted in fulfilment of the requirements for

the degree of Magister Scientiae at the Nelson

Mandela Metropolitan University

December 2012

Supervisor: Prof V. Oosthuizen

Co‐Supervisors: Dr S Moore and Prof. B Zeelie Abstract

False codling moth is a known pest of economic importance to many cultivated crops in South Africa and Africa south of the Sahara, and is particularly severe on citrus. If the fruit is infested just before harvest the chances of detecting signs of infestation are very low. As a result, the risk of packaging infested fruit and exporting them as healthy fruit is high. It is therefore a priority to develop a post-harvest technique for detection of False codling moth in citrus fruit at different levels of infestation in order to reduce phytosanitary risk. Compounds released and detected were indicative of infestation and were not insect produced but naturally produced fruit volatiles emitted at higher levels as a result of the insect within the fruit. Five major volatile compounds of interest were released by the infested oranges. These major volatile compounds include D-limonene, 3,7-dimethyl-1,3,6-octatriene, (E)-4,8-dimethyl- 1,3,7-nonatriene, caryophyllene and naphthalene. Limonene was one of the most abundant volatile compounds released by the infested citrus fruit. Naphthalene, which is possibly produced due to larval feeding and development within the fruit maintained higher concentrations than controls throughout the infestation within the fruit. Naphthalene would be a good indicator of False codling moth infestation, however, not primarily for early infestation detection. A significantly higher concentration of D-limonene, 3,7-dimethyl-1,3,6-octatriene, (E)-4,8-dimethyl-1,3,7- nonatriene and naphthalene was detected using the SEP over the SPME technique. The application of an SPME procedure and the utilization of this method for detection of volatiles present in the headspace of intact infested fruit are evaluated and the possible volatile compounds diagnostic of Thaumatotibia leucotreta infestation of orange fruit and differences in volatile compound response in different orange varieties is discussed.

MSc dissertation, page 1 DEPARTMENT OF ACADEMIC ADMINISTRATION

EXAMINATION SECTION

SUMMERSTRAND NORTH CAMPUS

PO Box 77000

Nelson Mandela Metropolitan University

Port Elizabeth

6013

Enquiries: Postgraduate Examination Officer

DECLARATION BY CANDIDATE

NAME: Rachel van der Walt

STUDENT NUMBER: 207036106

QUALIFICATION: Magister Scientiae Biochemistry

TITLE OF PROJECT: Identifying volatile emissions associated with False codling moth infested citrus fruit

DECLARATION:

In accordance with Rule G4.6.3, I hereby declare that the above-mentioned dissertation is my own work and that it has not previously been submitted for assessment to another University or for another qualification.

SIGNATURE: ______

DATE: ______

MSc dissertation, page 2 Acknowledgements

To my supervisor, Vaughan Oosthuizen, for his patience, assistance and for being so willing to take me on as a student.

I would like to extend a big thanks to Sean Moore for his guidance and for giving me the opportunity to take on a project which has so much potential and interest in the industry.

Thank you to my co- supervisor, Ben Zeelie, and associates at Innoventon who have assisted me and for being so willing to make use of the systems in the lab. I would like to acknowledge Cecilia Saunders and Melissa Gouws for their continuous assistance with the GC/MS and interest in my work which has kept me motivated.

To Coos Bosma for his help with the statistics and Ben Burger for sharing his knowledge on gas chromatography and great advice on using a SEP.

Big thanks to John Opoku-Debrah, Eugene Nepgen and Wayne Kirkman for technical assistance for this project.

To River Bioscience (Pty) Ltd. for providing False codling moth egg sheets for this project.

To the citrus growers at Sundays River Research Station (ARC, ITSC), Johan and Gideon, for allowing me to collect fruit and infest trees in their orchards.

To my family for their unconditional love and support.

Thank you to Jeffrey Kennedy for his never ending support and encouragement.

Thank you to the following organizations for their financial support: Citrus Academy, Citrus Research International and Nelson Mandela Metropolitan University.

MSc dissertation, page 3

Table of Contents

Abstract……………………………………………………………………………………… 1

Declaration………………………………………………………………………………….. 2

Acknowledgements………………………………………………………………………… 3

Table of Contents………………………………………………………………………….. 4

List of Abbreviations……………………………………………………………………….. 7

List of Figures………………………………………………………………………………. 8

List of Tables……………………………………………………………………………… 11

Chapter 1

1. Literature Review...... 14

1.1. Citrus cultivation in South Africa...... 14

1.2. False codling moth...... 14

1.2.1. Life cycle...... 15

1.2.2. Distribution...... 15

1.2.3. Effects on cultivation...... 17

1.3. Volatile compounds released from fruit...... 18

1.3.1. Fruit volatile compounds associated with insect infestation...... 19

1.3.2. Volatiles released by citrus fruit...... 20

1.4. Volatile analysis from headspace...... 22

1.4.1. Solid Phase Micro-extraction...... 22

1.4.2. Alternative approaches…...... 24

MSc dissertation, page 4 1.4.3. Sample enrichment probe...... 26

Chapter 2

2. Introduction...... 28

2.1. Justification...... 28

2.2. Aims and Objectives…...... 29

Chapter 3

3. Detection of headspace volatiles surrounding False codling moth infested citrus fruit……………………………………...... 30

3.1. Methods and Materials...... 30

3.1.1. Plant material, insect infestation and storage conditions...... 30

3.1.2. Volatile collection using Solid Phase Micro-extraction...... 31

3.1.3. Volatile collection analysis using GC-MS...... 32

3.1.4. Comparison of volatile collection using a sample enrichment probe and Solid Phase Micro-extraction...... 32

3.1.5. Data Analysis...... 33

Chapter 4

4. Results...... 35

4.1. Volatile compounds detected using SPME-GC/MS from False codling moth larvae infested Navel and Valencia oranges (Citrus sinensis L. Osbeck)...... 37

4.2. Major volatile compounds detected using the SPME-GC/MS and non- parametric analysis...... 37

4.2.1. Forcibly infested early harvest Cara cara Navel oranges...... 37

4.2.2. Forcibly infested late harvest Lane late Navel oranges...... 43

MSc dissertation, page 5

4.3. Major volatile compounds detected using the SPME-GC/MS from naturally infested late harvest Lane late Navel and late harvest Midnight Valencia oranges ...... 49

4.4. Major volatile compounds emitted from naturally infested Midnight Valencia oranges using SPME-GCMS and SEP procedures...... 53

Chapter 5

5. Discussion...... 57

5.1. Conclusions...... 64

5.2. Future work...... 66

Chapter 6

6. Appendix...... 68

Chapter 7

7. References...... 74

MSc dissertation, page 6 List of Abbreviations

FCM False codling moth GC Gas chromatography GC/MS Gas chromatography/Mass spectrophotometry HCSP High-capacity sorption probe p Probability PDMS Poly dimethyl siloxane PPT Parts per thousand SBSE Stir-bar-sorptive extraction SEP Sample enrichment probe SPACE Solid-phase aroma-concentrate extraction SPME Solid Phase Micro-extraction TR Treatment TD Thermal desorption µg/ml Micrograms per millilitre

MSc dissertation, page 7 List of Figures

Chapter 1

Figure 1: Geographic distribution of False codling moth...…...... 17

Figure 2: Relationship between different SPME fibres and target analyte molecular weight range …………………………………..……………………………………...... 22

Figure 3: SPME collection procedure. Piercing the sample septum; exposing the fibre to analytes and the removal of the fibre from the enclosure……………………………………………………………………...…………... 23

Figure 4: SPME analysis procedure. Piercing the GC inlet septum; exposing the fibre containing analytes for thermal desorption and the removal of the fibre from the enclosure …………………………………………………………………………………..24

Chapter 4

Figure 5: Box plot showing the median concentration of D-limonene released from the infected healthy and infested Cara cara Navel oranges for the experimental period between 18 and 25 days after infestation……………………………………....37 Figure 6: Box plot showing the median concentration of 3,7-dimethyl-1,3,6- octatriene released from the infected healthy and infested Cara cara Navel oranges for the experimental period between 18 and 25 days after infestation……………...38 Figure 7: Box plot showing the median peak area of (E)-4,8-dimethyl-1,3,7- nonatriene released from the infected healthy and infested Cara cara Navel oranges for the experimental period between 18 and 25 days after infestation……………...39 Figure 8: Box plot showing the median peak area of caryophyllene released from the infected healthy and infested Cara cara Navel oranges for the experimental period between 18 and 25 days after infestation……………………………………………….40 Figure 9: Box plot showing the median concentration of naphthalene released from the infected healthy and infested Cara cara Navel oranges for the experimental period between 18 and 25 days after infestation……………………………………….41 Figure 10: Box plot showing the median concentration of D-limonene released from the infected healthy and infested Lane late Navel oranges for the experimental period between 18 and 25 days after infestation……………………………………….43 Figure 11: Box plot showing the median concentration of 3,7-dimethyl-1,3,6- octatriene released from the infected healthy and infested Lane late Navel oranges for the experimental period between 18 and 25 days after infestation………………44 Figure 12: Box plot showing the median peak area of (E)-4,8-dimethyl-1,3,7- nonatriene released from the infected healthy and infested Lane late Navel oranges for the experimental period between 18 and 25 days after infestation………………45 Figure 13: Box plot showing the median peak area of caryophyllene released from the infected healthy and infested Lane late Navel oranges for the experimental period between 18 and 25 days after infestation……………………………………….46

MSc dissertation, page 8 Figure 14: Box plot showing the median concentration of naphthalene released from the infected healthy and infested Lane late Navel oranges for the experimental period between 18 and 25 days after infestation……………………………………….47

Figure 15:Effect of False codling moth larvae infestation of Lane late Navel and Midnight Valencia oranges on the concentration (µg/ml) of D-limonene after natural infestation within the orchard ……………………………………………………...….....48

Figure 16: Effect of False codling moth larvae infestation of Lane late Navel and Midnight Valencia oranges on the concentration (µg/ml) of 3,7-dimethyl-1,3,6- octatriene after natural infestation within the orchard ……………………………..…..49

Figure 17: Effect of False codling moth larvae infestation of Lane late Navel and Midnight Valencia oranges on the concentration (µg/ml) of (E)-4,8-dimethyl-1,3,7- nonatriene after natural infestation within the orchard ………………………..……....50

Figure 18: Effect of False codling moth larvae infestation of Lane late Navel and Midnight Valencia oranges on the concentration (µg/ml) of caryophyllene after natural infestation within the orchard ………….…………………………………...…...51

Figure 19: Effect of False codling moth larvae infestation of Lane late Navel and Midnight Valencia oranges on the concentration (µg/ml) of naphthalene after natural infestation within the orchard ………………………………..…………………...……...52

Figure 20: Comparison of the effect of False codling moth larvae infestation of Midnight Valencia oranges on the response of A: D-limonene; B: 3,7-dimethyl-1,3,6- octatriene, C: (E)-4,8-dimethyl-1,3,7-nonatriene; D: caryophyllene; and E naphthalene using a standardized SPME-GC/MS and SEP-GC/MS procedure, after natural infestation within the orchard. Bars with the same letter do not differ significantly by Mann-Whitney test (p = 0.05)...... 55

Chapter 6

Figure 21: Limonene standard concentrations and their respective peak areas used for quantification………………………………………………………………………...... 67

Figure 22: 3,7-dimethyl-1,3,6-octatriene standard concentrations and their respective peak areas used for quantification………………………………………....68

Figure 23: Naphthalene standard concentrations and their respective peak areas used for quantification…………………………………………………………………....68

Figure 24: Chromatogram showing the peak height, peak area and retention time for 1 µg/ml of D-Limonene commercial standard used for quantification...... 69

Figure 25: Chromatogram showing the peak height, peak area and retention time for 1 µg/ml of 3,7-dimethyl-1,3,6-octatriene commercial standard used for quantification...... 70

MSc dissertation, page 9 Figure 26: Chromatogram showing the peak height, peak area and retention time for 1 µg/ml of naphthalene commercial standard used for quantification...... 71

Figure 27: Typical chromatogram from the healthy fruit samples showing the peak height, peak area and retention time of the volatiles found in the headspace of intact oranges...... 72

Figure 28: Typical chromatogram from the infested fruit samples showing the peak height, peak area and retention time of the volatiles found in the headspace of intact oranges...... 73

MSc dissertation, page 10 List of Tables

Chapter 1

Table 1: Concentration (µg/ml) of the major volatile compounds quantified in Valencia and Navel oranges as well as literature confirming these volatile compounds using the SPME-GC/MS technique ……………………………………….21

Chapter 2

Table 2: Normality test statistics for volatiles emitted from Cara cara Navel oranges for both healthy and infested fruit over a period of 25 days ……………………….....33

Table 3: Normality test statistics for volatiles emitted from Lane late Navel oranges for both healthy and infested fruit over a period of 25 days .…………………………33

Chapter 4

Table 4: Concentration (µg/ml) of the volatile compounds detected in False codling moth larvae infested Valencia and Navel oranges using the SPME-GC/MS detection technique…...... 35

Table 5: Median and Mann-Whitney test values for the response of D-limonene, 3,7- dimethyl-1,3,6-octatriene, (E)-4,8-dimethyl-1,3,7-nonatriene, caryophyllene and naphthalene in healthy and infested Cara cara Navel oranges for the experimental period between 18 and 25 days after infestation…..………………………………….36 Table 6: Median and Mann-Whitney test values for the response of D-limonene, 3,7- dimethyl-1,3,6-octatriene, (E)-4,8-dimethyl-1,3,7-nonatriene, caryophyllene and naphthalene in healthy and infested Lane late Navel oranges for the experimental period between 18 and 25 days after infestation……………………………………...42

MSc dissertation, page 11 Chapter 1

1. Literature Review

1.1. Citrus cultivation in South Africa

In 1998 South Africa had over 18 million citrus trees covering a total area of more than 47 422 hectares (Bedford, 1998). The export of citrus from South Africa, together with Mozambique, Swaziland and Zimbabwe produced export revenue of approximately R 1500 million per annum (Bedford, 1998). After the 2011 season South African citrus production had an annual value of R46 206 329 according to Citrus Grower Association Annual Report 2012. The major citrus producing areas in South Africa include the Northern Province, Mpumalanga as well as the Eastern and Western Cape provinces. The Eastern Cape consists of four main citrus localities, with the lower Sundays River Valley and Gamtoos River Valley rated as the major producers (Bedford, 1998).

1.2. False codling moth

Climatic conditions are variable between different citrus areas and also within these areas which results in differences in the distribution of pests. This is due to the fact that the pests have varying sensitivity to temperature and humidity (Newton, 1998). An important citrus pest in all of the major citrus areas of South Africa is the false codling moth (Thaumatotibia leucotreta Meyrick), which has quickly invaded areas that are abundant in citrus due to an increase in commercial plantings (Newton, 1998). The false codling moth is in the Tortridae family under the order of Lepidoptera, which has some of the most important pests of Insecta (Pedigo and Rice, 2008). Lepidoptera is one of the largest orders, with a total of 150 000 species, of which 10 000 to 12 000 are found in South Africa (Scholtz and Holm, 1985). The adults live almost entirely on nectar and over-ripe fruit, while the larvae possess

MSc dissertation, page 12 chewing mouthparts that allow them to feed, with a few exceptions, entirely on plant parts (Scholtz and Holm, 1985).

1.2.1. Life cycle

The life cycle of the False codling moth, excluding diapause, consists of egg, larval, pupal and adult stages. The complete life cycle ranges from 30 days under favourable conditions, to 174 days in poor conditions (Stibick, 2010). Eggs of the False codling moth may be laid on or near the food source, singly or in batches. According to Bedford et al., (1998), in citrus the majority of eggs are laid directly on the fruit surface. Any larvae that feed inside the fruit must tunnel from the egg directly into it because the larval ovipositor is not able to penetrate the fruit (Scholtz and Holm, 1985). Egg development requires two to 22 days, depending on temperature (Stibick, 2010). After hatching, the 1 mm long, spotted larvae gnaw a burrow 1 mm into the rind of the host plant’s fruit. Emerging larvae usually feed just below the fruit surface or occasionally tunnel through the pith to the core of the fruit (Newton, 1998). The entrance to this burrow is easily seen by the yellow-brown discolouration of the fruit around the point of infestation due to tissue decay (Newton, 1998). When the emerging larva penetrates the fruit this leads to fruit decay, premature ripening and abscission (Newton, 1998). Once larvae are in the pulp they begin to feed. Only a limited number of larvae are able to survive per an individual fruit. Even in cases of high larval mortality, the exploring larvae create small lesions, which on their own can lead to fruit loss (Newton, 1998). The larval period lasts 12 to 33 days and consists of five instars (Stibick, 2010). Once the larvae have matured they emerge from the fruit and drop to the ground on silken threads. On emergence the larvae have changed into a pink colour and are now 15 mm long. In laboratory culture many hundreds of larvae can be reared in single containers of artificial diet (Newton, 1998).

MSc dissertation, page 13 1.2.2. Distribution

False codling moth is known to occur in citrus of South Africa, Mozambique, Zimbabwe and Swaziland (Newton, 1998). It is native to Africa and it is found mostly in sub- Saharan Africa and the nearby islands in the Atlantic and Indian Oceans (Figure 1) (Newton, 1998). False codling moth is known to occur in a variety of countries outside of South Africa and associated with the African continent including: Angola, Benin, Burkina Faso, Burundi, Cameroon, Cape Verde, Central African Republic, Chad, Congo Democratic Republic, Cote d’Ivoire, Eritrea, Ethiopia, Gambia, Ghana, Israel, Kenya, Madagascar, Malawi, Mali, Mauritius, Mozambique, Niger, Nigeria, Réunion, Rwanda, Saint Helena, Senegal, Sierra Leone, Somalia, South Africa, Sudan, Swaziland, Tanzania, Togo, Uganda, Zambia, and Zimbabwe (Venette et al., 2003). In most equatorial areas False codling moth has infested cotton; in South Africa it predominates in citrus and has recently been found occurring in macadamia nuts in Malawi (Stibick, 2010). In a study done from 1984 to 2008 in the United States, 1500 occurrences of False codling moth were found on 99 different plant taxa at 34 different entry points (Venette et al., 2003). False codling moth has been recorded to feed on over fifty species of plants including Abelmoschus esculentus (okra), Ananas comosus (pineapple); Annona reticulata (custard apple), Camellia sinensis (tea), Capsicum annuum var. annuum (sweet pepper), Citrus aurantium (sour orange), Citrus paradisi (grapefruit), Citrus spp., Coffea arabica (arabica coffee), Diospyros virginiana (persimmon), Ficus sp. (wild fig), Garcinia mangostana (mangosteen), Gossypium sp. (cotton), Harpephyllum caffrum (plum), Hibiscus sp., Litchi chinensis (litchi), Mangifera indica (mango); Olea europaea (olive), Persea americana (avocado), Pimenta dioica (pimento), Piper sp. (pepper), Prunus domestica (plum, prune), Prunus persica (peach), Prunus persica var. nucipersica (nectarine), Psidium guajava (guava), Punica granatum (pomegranate), Quercus robur (English oak), Quercus spp. (acorn, oak), Ricinus communis (castor bean), Sorghum bicolor (sorghum) and Zea mays (maize) (van der Geest et al., 1991). Due to an uninterrupted supply of plant hosts, False codling moth remains active throughout the year (Stibick, 2010).

MSc dissertation, page 14

Figure 1: Geographic distribution of False codling moth in Africa. Bullets indicate the location where the pest has established itself (Stibick, 2010)

1.2.3. Effects on cultivation

False codling moth is a known pest of economic importance to many cultivated crops; it attacks many deciduous, subtropical and tropical fruits but it is particularly severe on citrus. Although in South Africa citrus seems to be the major host plant that is affected by False codling moth, it is also a major pest on macadamias in this region (Newton, 1998). The Navel orange seems to be the most severely attacked citrus cultivar, with more eggs being laid on this cultivar than any other (Newton, 1998). Temperature, food availability and quality, photoperiod, humidity, latitude and the effect of predators and diseases are known to influence the number of generations per year (Stibick, 2010). In addition to citrus fruit, False codling moth is also known as a pest of certain plant parts that are economically important particularly in South Africa. These include avocado fruit (Persea americana),

MSc dissertation, page 15 macadamia nuts (Macadamia spp.), peach and plum fruit (Prunus spp.), sacorns (Quercus spp.), walnuts (Junglans regia L.), olives (Olea europaea), tea seeds (Camellia sinensis) and almonds (Prunus dulcis Miller) (Newton, 1998; van der Geest et al., 1991).

Any stage of fruit development can be influenced by False codling moth larval feeding and development. Typically, this results in premature ripening and fruit drop (Stibick, 2010). False codling moth can cause significant crop losses and undetected infestation before harvest can cause decay of fruit once it has been packed and shipped to export markets (Rogers et al., 2007). The United States Department of Agriculture estimated that crop yield losses in some cases were as high as 20% (Stibick, 2010). False codling moth is a major problem to the citrus industry in both South Africa and other citrus producing countries. In South Africa, citrus crop losses due to False codling moth infestation have been reported to be around 10-20% of the crop yield (van der Geest et al., 1991). A 42-90% decrease in crop production of macadamias in Uganda and a 28% decrease in South African production of peaches has been reported (van der Geest et al., 1991). False codling moth is a difficult pest to control as eggs are laid continuously during the fruiting period of citrus (Newton, 1998). Once the fruit has been penetrated, it is no longer marketable. Chemical control of False codling moth is difficult because of the inaccessibility of many of the life stages, the capacity of the moth to develop resistance to chemical insecticides, and high cost due to the persistent pest pressure throughout the fruiting season (Newton, 1998). Effective control of False codling moth is therefore difficult to achieve and insight into the prevention of the spread of infestation is encouraged.

1.3. Volatile compounds released from fruit

Plants release volatile compounds resulting from metabolic activities taking place within the plant parts such as shoots, leaves, flowers or fruits (Sankaran et al., 2010). The volatile compound profile of infested plant parts is significantly different due to the physiological condition of the plant as well as the species or the cultivar

MSc dissertation, page 16 (Sankaran et al., 2010). The volatile compound profile of a particular plant can be influenced by changes in the plant’s metabolism due to alterations in the environment, the age of the plant, the development stage of the plant, effect of different stresses on the plant, as well as the presence of disease or herbivores in the plant parts (Sankaran et al., 2010). The use of volatile plant metabolites as an indicator of disease, stress or herbivore presence is challenged by the natural variation, within plant species, of the volatile compound profile (Sankaran et al., 2010). It is vital to correctly identify a distinct volatile compound which can act as a biomarker for a particular plant and herbivore which will be different from a volatile compound produced due to environmental or nutrient stress (Sankaran et al., 2010).

Only recently have there been advances in sensitive chemical technology which have been successfully applied postharvest for the detection of insects (Kendra et al., 2011). Examples include the detection of stink bugs and bollworm damage in cotton, the detection of grain borers and the feeding damage of grain borers in wheat (Kendra et al., 2011). The identification of food infestation associated volatile compounds can be exploited which will allow for improvement of pest detection in the future.

1.3.1. Fruit volatile compounds associated with insect infestation

The expected differences in volatile emission between False codling moth infested, healthy mechanically injured fruit, and healthy fruit are either release of different types of volatile compounds, or release of different relative amounts of volatile compounds. Another possible expectation would be the release of different compounds with time within the infested fruit due to larval development over time.

The possible production of novel compounds due to infestation of plant parts has been indicated by previous research conducted on Walnut husks where three volatile compounds were emitted by codling moth infested husks and not from green or overripe husks (Buttery et al., 2000). Staudt and Lhoutellier (2007) also found that the leaves of the holm oak tree infested by gypsy moth larvae released

MSc dissertation, page 17 at least five volatiles which were not released by the healthy leaves. Both Boeve et al., (1996) and Landolt et al., (2000) have shown that apples in response to attack by European apple sawfly produce (E,E)-α-farnesene along with additional volatile chemicals which is thought to attract additional insects. Different relative amounts of volatile compounds have also shown to be indicative of infestation of plant parts in previous studies. Boeve et al., (1996) found that although apples infested with European apple sawfly larvae emitted the same volatiles as healthy apples, three of the terpinoids detected were emitted in significantly larger amounts. In some cases, such as the infestation of silver birch with herbivore arthropod Epirrita autumnata, it was shown that several volatile compounds were elevated with infestation as well as having an additional volatile emitted due to infestation. Recently, Kendra et al., (2011) found that mangos infested with Anastrepha ludens not only released higher amounts of specific volatiles but also released additional volatiles that were not present in the healthy controls. Kendra et al., (2011) also found that the increased release of hexyl butanoate from citrus which was induced by infestation by Caribbean fruit fly Anastrepha suspensa (Loew) was highest during the first instar. This initial stage of infestation is the most difficult to detect by visual inspection, and levels declined with subsequent instars. This suggests that larval development and their activities, or lack thereof, within the fruit can influence the production of different volatiles. This pattern of induced volatile emissions has been documented by Hern and Dorn (2001) where apples infested with larvae of the codling moth (Cydia pomonella L.) emitted high levels of volatiles when the larvae was at its first instar within the infested fruit. However, within 9-12 days the amounts decreased to levels equivalent to that from healthy fruits. In contrast, Boeve et al., (1996) found that when apples were infested with European apple sawfly larvae, 2-phenylethanol was found in larger amounts during late infestation.

MSc dissertation, page 18 1.3.2. Volatiles released by citrus fruit

The most predominant volatile compounds detected in the atmosphere surrounding wounded and infested fruit of different citrus species include the monoterpenes which include limonene, pinene, sabinene and myrcene (Droby et al., 2008). Monoterpene hydrocarbons are typically the most abundant volatile compounds in all volatiles regardless of the condition of the source (Droby et al., 2008). The emission of volatile compounds from healthy ripe grapefruit (Citrus paradisi L.) was shown to consist almost exclusively of monoterpene hydrocarbons (Flamini et al., 2010). The most abundant volatile compound in citrus, limonene, was shown to increase from 34% to 95% of total emissions as the fruit ripened (Flamini et al., 2010). The volatile profile of citrus fruit differs between species but the same cannot necessarily be said between cultivars with a species (Birla et al., 2005). The cited concentration range of the 16 most predominant volatiles associated with Citrus sinensis fruit from both Navel and Valencia cultivars are given (Table 1). This is supported by literature which confirmed presence of these volatile compounds in either Navel or Valencia oranges.

Table 1: Concentration (µg/ml) of the major volatile compounds quantified in Valencia and Navel oranges as well as literature confirming these volatile compounds using the SPME-GC/MS technique

Valencia and Volatile Navels LV compound (μg/mL) Cited in Literature Acetaldehyde 3-8.5a Birla et al., 2005 Ethanol 64-900a Birla et al., 2005 Ethyl acetate 0.01-0.58b Birla et al., 2005 Hexanal 0.02-0.65b Birla et al., 2005 Ethyl butanoate 0.26-1.02b Birla et al., 2005 Droby et al., 2008; Flamini et al., 2007; α-Pinene 0-0.22b Birla et al., 2005 Droby et al., 2008; Flamini et al., 2007; Sabinene 0-0.15b Birla et al., 2005 Droby et al., 2008; Flamini et al., 2007; β-Myrcene 1.54c Birla et al., 2005 Droby et al., 2008; Flamini et al., 2007; Limonene 1-278b Birla et al., 2005 1-Octanol NA* Birla et al., 2005 λ-Terpinene 0.04-0.46a Flamini et al., 2007

MSc dissertation, page 19 Linalool 0.15-4.6a Droby et al., 2008; Birla et al., 2005 L- α -Terpineol 0.09-1.1a Birla et al., 2005 Decanal 0.01-0.15a Droby et al., 2008; Birla et al., 2005 Dodecanal NA Birla et al., 2005 Valencene 0.8-15b Birla et al., 2005

NA, not available; *, not detected in Navel oranges; LV, literature values cited from: (a) Shaw (1986), (b) Nisperos-Carriedo and Shaw (1990), (c) Steffen and Pawliszyn (1996) within Birla et al., 2005

1.4. Volatile analysis from headspace

1.4.1. Solid Phase Micro-extraction

The analysis of volatile compounds by static headspace analysis is widely used (Miller and Stuart, 1999). However, in many analyses, the gas-sampled static headspace method using an air tight syringe lacks the sensitivity required (Miller and Stuart 1999). Solid phase micro extraction (SPME) can perform analyses at a level of sensitivity that gas sampling is unable to achieve (Miller and Stuart, 1999). SPME was developed by Arthur and Pawliszyn in 1990 and is used for quantitative trace analysis of volatile and semi-volatile compounds (Pawliszyn, 1999).

SPME technique allows for good selectivity, as different adsorption fibres can be chosen depending on the target compound to be analyzed (Wang 1997). SPME fibre coatings have different chemical properties but the most popular SPME fibres are made of a polydimethylsiloxane stationary phase (Wang, 1997). By choosing the correct fibre for the analyte to be collected, the sensitivity of the analysis can be improved (Figure 2) (Wang, 1997).

MSc dissertation, page 20

Figure 2: Relationship between different SPME fibres and target analyte molecular weight range. TPR:Templated resin; PDMS: Polydimethylsiloxane; DVB: Divinylbenzene (Mindrup and Shirey 2001)

The SPME extraction principle is described as an equilibrium process in which the volatile analytes are partitioned between the stationary phase of the fibre and headspace (Wang, 1997). SPME allows solventless extraction by using a fused silica or stainless steel fibre coated with a thin polymer. This acts as a solvent and allows the compounds to be extracted (Pawliszyn, 1999). The analytes absorb to the polymer coating on the SPME fibre, which is mounted onto a syringe like device (Figure 4) (Wang, 1997). Once the SPME fibre is placed in the GC inlet the analytes are thermally desorbed almost instantaneously and are transferred onto the column of the gas chromatograph for separation and further quantification (Pawliszyn, 1999).

MSc dissertation, page 21

Figure 3: SPME collection procedure. Piercing the sample septum; exposing the fibre to anayltes and the removal of the fibre from the enclosure (Mindrup and Shirey 2001)

MSc dissertation, page 22

Figure 4: SPME analysis procedure. Piercing the GC inlet septum; exposing the fibre containing analytes for thermal desorption and the removal of the fibre from the enclosure (Mindrup and Shirey, 2001)

SPME is able to combine extraction, concentration and introduction of volatile analytes in one step which reduces preparation time and in some cases this fact increases sensitivity over other extraction techniques (Pawliszyn, 1999). It follows that the main advantage of SPME over other techniques is that the commercially available SPME devices are convenient and user friendly.

1.4.2. Alternative approaches

SPME has its benefits over other reliable methods, however, the SPME procedure also results in low enrichment due to the poor partitioning of the very small volumes (microlitre) of the sorbent on the fibre and the large sample volumes (Burger et al.,

MSc dissertation, page 23 2006). Other approaches that allow enrichment of headspace volatile compounds for analysis can be achieved using stir-bar-sorptive extraction (SBSE), solid-phase aroma-concentrate extraction (SPACE) or a high-capacity sorption probe (HCSP). SBSE was invented several years after SPME by Pat Sandra and colleagues, with the main advantage of vast sensitivity (Pawliszyn, 1999). SBSE is able to be used for analysis of gas or liquid samples by making use of a sorptive sleeve of rubber on a magnetic follower or stir-bar which is placed within the sample to extract the analyte (Vas and Key, 2004). The volume of the sorptive stationary phase on a SBSE stir- bar can be increased beyond that of the commercially available stir-bars. The main disadvantage of SBSE is the requirement of a highly expensive thermal desorption/cryotrapping unit that is required to desorb the volatiles, condense them and transport them into the column as a narrowly focused band. Desorption of volatiles is not instantaneous when using SBSE however, this technique is more than 50 times more sensitive than SPME. SBSE requires the use of a relatively large sorptive volume of stationary phase which results in a greater degree in sensitivity of volatile detection than SPME (Vas and Key, 2004). Generally SBSE procedures are not utilized for many different applications requiring extraction procedures; however, SBSE has a greater capacity for quantitative extraction than SPME (Mitra, 2004). Much like SBSE, extraction of analytes using HCSP has the advantage of having increased sensitivity with larger amounts of sorptive stationary phase (Mitra, 2004). HCSP is able to extract large amounts of analyte from samples, however, selectivity of this extraction technique is low (Burger et al., 2006). SPACE is a more recently introduced sample enrichment method where volatile compounds are enriched on a graphite- carbon absorbant on a stainless steel rod (Burger et al., 2006). The primary advantage of SBSE, SPACE and HCSP, using large amounts of PDMS sorbent material to achieve greater concentration capabilities, can also be their greatest disadvantage compared with SPME fibres (Mitra, 2004). The non-selective sorptive capability of the PDMS fibre does not allow for selection of a particular sized volatile compound of interest and could result in the concentration of undesirable matrix components during the absorption phase of the procedure (Mitra, 2004). Although high sensitivity is achieved using SBSE, SPACE and HCSP through use of a larger sorptive material, these methods require a thermal desorption procedure and volatiles contained on these fibres require cryofocusing onto the column (Burger et al., 2006). The large volumes of sorptive material used in SBSE, SPACE and HCSP

MSc dissertation, page 24 rule out instantaneous desorption of the volatiles, and therefore have to be cryofocused on the column (Burger et al., 2006). These high sensitivity techniques require cryofocusing and desorption of volatiles onto the column which results in the need for additional expensive systems and conversions. SPME and SEP do not require cryofocusing of volatiles onto the column and thermal desorption occurs almost immediately after injection of the sample into the chromatography system, which eliminates the requirement for a thermal desorption system (Burger et al., 2011).

1.4.3. Sample enrichment probe

SPME makes use of a very small volume of sorptive phase. Absorption of volatiles onto the fibre is allowed to proceed until the rate of adsorption is equal to the rate of release. Heat is able to change this equilibrium to allow the concentrated volatiles to be released, therefore thermal desorption of the enriched material is required (Burger et al., 2006). The sample enrichment probe (SEP) technique differs from the SPME in that a larger volume of the sorptive phase is utilized; the sorptive phase is not introduced into the inlet of the GC via a needle. In addition, the use of the SEP technique does not require any thermal desorption unit or cryotrapping equipment. However, it may require minor adaptations to the gas chromatograph inlet system for insertion of the sample into the unit (Burger et al., 2006). The analytes captured on this probe are desorbed in the injector of the gas chromatograph and cryotrapping is thus circumvented (Burger et al., 2011). The SEP is made of stainless steel and a 15 mm rubber sleeve. The SEP technique makes use of relatively large amounts of PDMS rubber in order to produce results that in principle should be comparable with SBSE and SPME (Burger et al., 2011). The sensitivity of SEP is the same as that of SBSE if the same volume of sorptive phase is used on the stir-bar and the stalk of the SEP. Desorption is not instantaneous, and because the desorption temperature is about 220°C-230°C in comparison to the 300°C, desorption is slower in SBSE. The fact that this technique is capable of producing results similar to that of SBSE can be explained as follows: The highly volatile analytes are desorbed instantaneously, are transported into the column by the carrier gas and are eluted as sharp peaks.

MSc dissertation, page 25 However, desorption of the higher compounds starts at the same time but desorption continues for some time after the analysis has started or even after volatiles have reached the detector. The broadening and tailing of peaks of the higher volatiles undergo cold trapping which refers to the trapping on the column of molecules that have low vapour pressures and therefore don’t migrate at low column temperatures. Therefore, the volatile compounds are desorbed rapidly and undergo gas chromatographic separation while the less volatile analytes are slowly desorbed and gradually accumulate in the first few centimetres of the column until the temperature rises and they migrate. In contrast to other techniques, the injector of the GC is open to the atmosphere for a few seconds when the SEP is introduced. The injector has to be closed as rapidly as possible to restrict losses of the most volatile of the analytes (Ben Burger Pers. Comm). According to Burger et al., (2011) since the introduction of the SEP, this technique has shown to produce good qualitative results for several applications in the laboratory. Low cost and simplicity of the equipment used are advantages of the SEP technique over SBSE and SPME. However, the method is extremely particular and the procedure must be carried out exactly as stated or else results will not be accurate (Ben Burger Pers. Comm).

MSc dissertation, page 26 Chapter 2

1. Introduction

1.1. Justification

Control of False codling moth is challenging as eggs are laid continuously during the fruiting period of citrus. Effective methods for control of False codling moth on citrus pre-harvest exist. However; none of these techniques are able to gain 100% control. Once the fruit has been penetrated by the larvae of the moth, it is no longer marketable. False codling moth is a phytosanitary pest for a number of South African export markets, which may have a zero-tolerance for infested fruit. If the fruit is infested just before harvest, it is difficult to detect. Therefore, there is a risk of packaging infested fruit and exporting them as healthy fruit. It is therefore a priority to develop a post-harvest technique for detection of False codling moth in citrus fruit at different stages of False codling moth infestation in order to reduce phytosanitary risk. Using gas chromatography coupled to mass spectrophotometry to identify volatile compounds or patterns which are uniquely associated with False codling moth infested citrus fruit will be a major breakthrough in the detection of infestation in citrus, provided such a method can be reduced to practice during post-harvesting processing of the fruit.

This study will determine whether volatile analysis can be utilized as early detection of False codling moth larvae within intact fruit in cases where False codling moth is not completely controlled within orchards. The outcome of this project will assist citrus growers in making decisions about their pest management programs as well as contribute toward the development of a detection system which will immediately show infested fruit while on a packing line. Further research could later be focused on determining a more affordable or appropriate technology to confirm volatile presence within infested fruit postharvest.

MSc dissertation, page 27

1.2. Aims and Objectives

The aims of this project were to:

- Develop a volatile detection system for use on citrus fruit and; - Use this system to investigate differences in volatile emission between infested and healthy fruit.

The main objectives were to establish a comprehensive range of the various volatiles emitted by citrus fruit, to determine the differences in volatile emission by infested and non-infested citrus fruit, to determine differences in volatile emission by fruit infested at various intervals before testing and to establish differences in volatile emission by fruit as per life-stage of the larvae infesting the fruit. The differences in volatile emission by citrus fruit of different cultivars and varieties were also determined. The ultimate outcome being that this will lead to the development of a detection system in the future which will immediately show infested fruit while on a packing line.

MSc dissertation, page 28 Chapter 3

3. Detection of headspace volatiles surrounding False codling moth infested citrus fruit

3.1. Materials and Methods

3.1.1. Plant material, insect infestation and storage conditions

Forcibly infested fruit included Cara Cara’s, an early harvest variety of Navel oranges (Citrus sinensis L. Osbeck), as well as an early harvest variety of Valencia oranges. Naturally infested late variety Lane Late Navel oranges were utilized due to low temperatures and unfavourable weather conditions for penetration of neonate larvae of false codling moth. Naturally infested Midnight Valencias were analysed, allowing for the comparison of both naturally and force infested Navel and Valencia oranges.

Orange fruit on orchard trees were force infested with False codling moth larvae two weeks before the expected harvest date. Oranges chosen for infestation were inspected for uniformity as well as absence of any form of defects such as decay or external injuries. False codling moth eggs laid on squares of wax paper were glued onto each fruit ensuring a penetration of approximately three larvae per fruit. All False codling moth treated and control fruit was untreated, unwashed, and unwaxed. Fruit was then harvested at intervals after infestation to coincide with different stages of larval development. Fruit collection was carried out at intervals of 0, 5, 10, 18 and 25 days after infestation in order to coincide with any of the first instar, second instar and mid-third instar of the insects. At each interval 16 fruit samples were collected for the treatment as well as sampling 16 fruit for the non- infested control.

MSc dissertation, page 29 Chemical sampling was ideally done within 2 hours of harvesting. However, until the time of chemical sampling fruit was kept at a constant temperature of 20°C. This took into consideration the fruit storage, the insect rearing conditions as well as maintenance of the amount and type of volatiles (Bai et al., 2011). The optimum False codling moth rearing conditions are said to be not lower than 10°C and optimally at 27°C (Kendra et al., 2011). The optimal storage temperature for oranges is between the optimal temperature of 5°C and 25°C (Birla et al., 2005; Droby et al., 2008). This is due to oranges, a subtropical fruit, being sensitive to low temperatures of 4°C and below resulting in chilling injury which is associated with decreased aroma volatile production (Baldwin, 2004) as well as heat treatments above 25-30°C resulting in decreased volatile production (Baldwin, 2004). Chemical analyses were done at the same time of the day in order to avoid the influence of possible circadian rhythms and/or the influence of external stimuli such as light (Flamini et al., 2007). Following chemical collection and analysis, each fruit sampled was cut open and examined to determine the larval instar (first, second, third) and, by counting the number of larvae found within the fruit, estimate the level of insect infestation.

3.1.2. Volatile collection using Solid Phase Micro-extraction

An individual fruit was placed in a 3 L glass jar with Teflon septum fitted caps and allowed to equilibrate for 30 minutes at 30°C. Collection and trapping of headspace volatiles from the intact fruit was done using Solid Phase Micro-extraction (SPME). Each sample was collected by exposing a 100 µm polydimethyl siloxane (PDMS) non-bonded fibre (Supelco Co., Bellefonte, PA, USA) through the septa in the fruit jar to volatiles for 10 min of adsorption, after optimization of adsorption time. New fibres were used for control and infested fruit.

3.1.3. Volatile compound analysis using GC-MS

Gas chromatography/Mass spectrophotometry (GC/MS) analysis was done in order to analyze the volatile compounds from the head space of the infected and

MSc dissertation, page 30 uninfected fruit. GC/MS separation was done using an HP6890 series gas chromatograph (Agilent) equipped with a Zebron DB 5 (30 m, 0.25 mm internal diameter, 0.25 µm film thickness) capillary column and coupled to an HP 5973 mass selective detector (MSD). The temperature of the MSD was set at 230°C and the quadrapole set at 150°C. Chemicals absorbed by the SPME fibre were injected directly into the injector of the GC/MS by thermal desorption (TD) at 250°C for 2 min. Splitless injection was used with a 1 µL volume. Helium was used as a carrier gas at a constant flow rate of 0.05 ml.s-1. Prior to injection the oven was heated to 50°C, after injection the temperature increased at a rate of 0.167°C.s-1 until 130°C, followed by a 0.333°C.s-1 increase to 210°C which was held for 10 minutes. This temperature programme was modified from the method of Kendra et al. (2011) for optimum sensitivity of the probe in use. Scans were recorded for the mass range of 50–650 amu. Component peaks were identified using the NIST10/HPPest/Wiley275 mass spectral libraries and confirmed by comparing with the retention times of standard compounds. Quantification was achieved by comparing the peak areas of the samples to the peak areas of known concentrations of commercial standards (Figure 21; Figure 22 and Figure 23- Appendix). Standards were obtained from Sigma (St Louis, MO).

3.1.4. Comparison of volatile collection using sample enrichment probe and Solid Phase Micro-extraction

Three intact fruit were placed in a 3 L glass jar with Teflon septum fitted caps and allowed to equilibrate for 30 minutes at room temperature of approximately 22°C. Collection and trapping of headspace volatiles from the intact fruit was done using a sample enrichment probe (SEP) and an SPME probe for comparison. Each sample was collected by exposing a 100 µm polydimethyl siloxane (PDMS) non-bonded fibre through the septa in the fruit jar, to volatiles for 24 hours of sorption. The sampling was done in triplicate. GC separation was done using an HP5890 series gas chromatograph equipped with a DB 5 (30 m, 0.25 mm internal diameter, 0.25 µm film thickness) capillary column. The quadrapole was set at 180°C. Chemicals absorbed by the SEP and SPME fibres were injected directly into the injector of the GC by thermal desorption at 250°C for 2 min.Samples (1 µl) of standard solutions were

MSc dissertation, page 31 injected in the splitless mode. Helium was used as a carrier gas at a constant flow rate of 0.05ml.s-1. Prior to injection the carrier gas was manually switched off and the oven was kept as cool as possible. After placing the SEP fibre in the injection port/ injection of the SPME fibre, the carrier gas was switched on and the oven temperature initially increased to 40°C and then to 250°C at a rate of 4°C/min. This temperature programme was modified from the method of Kendra et al. (2011) for optimum sensitivity of the probe in use. Component peaks were identified using the NIST10/HPPEST/WILEY275 mass spectral libraries.

3.1.5. Data Analysis

A non parametric analysis of the data was proposed to detect any possible differences between the average difference in concentration of the emitted volatiles of the healthy and infested fruit over the experimental period of 25 days. This was applied to the force infested early harvest Cara cara and late harvest Lane late Navel oranges and their respective control fruit. The volatiles under consideration were: D- limonene, 3,7-dimethyl-1,3,6-octatriene, (E)-4,8-dimethyl-1,3,7-nonatriene, caryophyllene or naphthalene. Initial analysis showed that least squares regression to analyse the data is invalid. This is due to the responses not being normally distributed and containing many extreme outliers.

MSc dissertation, page 32 The results of the normality test on D-limonene, 3,7-dimethyl-1,3,6-octatriene, (E)- 4,8-dimethyl-1,3,7-nonatriene, caryophyllene and naphthalene for the different varieties analysed are shown in Tables 2 and 3.

Table 2: Normality test statistics for volatiles emitted from Cara cara Navel oranges for both healthy and infested fruit over a period of 25 days

Volatile compound Chi- df p-value Square

D-limonene 94.05 3 <0.0001

3,7-dimethyl-1,3,6-octatriene 84.85 8 <0.0001

(E)-4,8-dimethyl-1,3,7- nonatriene 574.7 6 <0.0001 caryophyllene 16.12 7 0.024 naphthalene 15.91 3 0.001

Table 3: Normality test statistics for volatiles emitted from Lane late Navel oranges for both healthy and infested fruit over a period of 25 days

Volatile compound Chi- df p-value Square

D-limonene 30.11 4 <0.0001

3,7-dimethyl-1,3,6-octatriene 44.24 4 <0.0001

(E)-4,8-dimethyl-1,3,7- nonatriene 47.97 3 <0.0001 caryophyllene 8.66 5 0.012

Naphthalene 37.81 1 <0.0001

MSc dissertation, page 33 It is clear from the chi-squared tests for normality and the associated p-values (Tables 2, and 3), that none of the volatile responses follow normal distribution. Transforming the data did not significantly improve the normality of the data.

An alternative approach to parametric regression analysis was made; non parametric statistics can be used to compare the median volatiles of the healthy and infested fruit at time intervals of the experimental period.

Statistical analysis for the comparison of emission of volatiles between healthy and infested data of the naturally infested late harvest Lane late Navel and late harvest Midnight Valencia as well as the comparison of volatile collection using sample enrichment probe and Solid Phase Micro-extraction consisted of an initial Shapiro- Wilks test showing that the data was not normal. The Mann-Whitney statistical test was used for statistical analysis as it does not depend on the normality of the data. All statistics were done using Statistica (StatsSoft Inc. Version 10).

MSc dissertation, page 34 Chapter 4

4. Results

4.1. Volatile compounds detected using SPME-GC/MS from False codling moth larvae infested Navel and Valencia oranges (Citrus sinensis L. Osbeck)

Volatile compounds which were released from the headspace of the majority but not all replicates sampled are shown in Table 4, indicating the concentrations (µg/ml) of these volatile compounds from early Navels, late Navels and late Valencias for each control, force infested and naturally infested samples. The volatile compounds released from and detected in all the replicates of the fruit sampled for both infested and non-infested fruit included D-limonene, 3,7-dimethyl-1,3,6-octatriene, (E)-4,8- dimethyl-1,3,7-nonatriene, caryophyllene and naphthalene. The changes in the concentration of hexyl butanoate in comparison to control fruit is also notable. The concentration of hexyl butanoate increased with the naturally infested late Navels (p = 0.008) and late Valencias (p = 0.005) as well as with the force infested early (p = 0.005) and late Navel fruit (p = 0.001).

MSc dissertation, page 35 Table 4: Concentration (µg/ml) of the volatile compounds detected in False codling moth larvae infested Valencia and Navel oranges using the SPME-GC/MS detection technique

Early Navels Late Navels Late Valencias Naturally Compound Control Force Infested Control infested Control Force Infested Control Naturally infested Acetaldehyde 0-0.01 0.01-0.06 0-0.01 0.01-0.06 0-0.01 0-0.01 0-0.01 0.06-0.13 2- propanol# 0-0.01 0.06-0.13 0.01-0.06 0.01-0.25 0.01-0.06 0.01-0.06 0.01-.25 0.06-0.13 2-ethyl-1-hexanol# 0-0.01 0.01-0.25 0-0.01 0-0.01 0-0.01 0-0.01 0-0.01 0-0.01 D-limonene^ 0.08-2.78 0.15-3.50+ 0.17-5.86 0.23-1.29 0.8-6.83 0.2-7.19 0.54-2.83 1.41-28.61+ 1,3,6 Octatriene 3,7 dimethyl (Z)^ 0.09-0.93 0.07-1.2+ 0.02-0.34 0.09-2.65 0.07-0.25 0.02-1.15* 0.24-3.26 1.22-16.56 1,6 Octadiene-3-ol 3,7 dimethyl# 0-0.01 0.01-0.5 0-0.13 0-0.25 0-0.13 0.01-0.06 0-0.01 0.01-0.06 Hexanoic acid# 0.01-0.06 0.25-1> 0.01-0.06 0.01-0.06 0.01-0.06 0.06-0.13 0.01-0.06 0.01-0.06 (E)- 4,8 Dimethyl 1,3,7 nonatriene# 0-0.13 0.06-0.25 0.01-0.25 0.01-0.25* 0.01-0.05 0.06-0.25 0.01-0.06 0.01-0.25* 1-Undecanol# 0-0.01 0.01-0.25 0.01-0.06 0.01-0.06 0.01-0.06 0-0.01 0.01-0.06 0-0.01 Hexyl butanoate# 0-0.01 0-0.5* 0-0.01 0.06-1>* 0.01-0.06 0.13-0.5* 0-0.01 0.01-0.5* Octanoic acid# 0-0.01 0-0.06 0-0.01 0.06-0.13 0-0.01 0.06-0.13 0-0.01 0-0.13 Dodecane# 0-0.01 0-0.06 0.06-0.13 0.25-1> 0.06-0.13 0.25-1> 0-0.01 0-0.01 2,6 Dihydroxyacetophenone bis (trimethylsilyl) ether# 0-0.01 0-0.06 0-0.01 0-0.01 0-0.01 0-0.01 0-0.01 0.01-0.06 Caryophyllene# 0.06-1 0.25-1 0.06-1 0.13-1 0.25-14 0.25-1* 0.01-0.06 0.06-0.5 Alloaromadendrene# 0.01-0.06 0-0.06 0 0 0 0 0.01-0.06 0.06-0.25 Humulene# 0-0.06 0.01-0.5 0 0 0 0 0-0.01 0.01-0.06 alpha.-Panasinsen# 0.06-0.25 0.01-1 0.01-0.06 0.06-0.5* 0.01-0.06 0.01-0.25 0.01-0.06 0.01-0.06 Naphthalene^ 0.05-0.14 0.01-1.28* 0.22-1.14 0-0.51 0.05-0.13 0.02-0.13* 0.123-0.785 0.266-6.30* 2-Isopropenyl-4a,8-dimethyl-1,2,3,4,4a,5,6,7- octahydronaphthalene# 0.25-0.5 0.5-1 0.25-0.5 0.25-1> 0.25-0.5 0.25-1> 0.25-0.5 0.25-0.5 Propanoic acid# 0.5-1 1> 0 0 0 0 0.01-0.06 0.13-0.25

Shaded rows indicate major volatile compounds found in the majority of samples. (^)= Quantified using commercially available standards; (#) = NIST mass spectral library (80> confidence level); (*) statistically significant different from control at p < 0.05; All concentrations in µg/ml

MSc dissertation, page 36 4.2. Major volatile compounds detected using the SPME-GC/MS and non- parametric analysis

4.2.1. Forcibly infested early harvest Cara cara Navel oranges

Initial analysis using scatter plots indicated that the only period where there was a possibility of a statistical difference between the responses of the healthy and infested fruit for all of the volatile compounds was after 18 days of infestation. The median values, number of replicates and Mann-Whitney statistics are shown in Table 5 for each of the volatile compounds of interest.

Median Median $ $ Mann Whitney Volatile compound N concentration concentration p-value healthy fruit infested fruit Test statistic Z D-limonene 32 1.178 0.504 0.345 0.118 3,7-dimethyl-1,3,6- 0.531 octatriene 32 0.038 0.067 0.049 (E)-4,8-dimethyl- 2.415 1,3,7-nonatriene 32 296144 259803 0.296 caryophyllene 32 1387689 1386778 1.794 0.714 naphthalene 23 0.272 0.264 -0.712 0.279

$; Concentrations for D-limonene, 3,7-dimethyl-1,3,6-octatriene, and naphthalene given in µg/ml; amounts for (E)-4,8-dimethyl-1,3,7-nonatriene and caryophyllene given as peak areas.

MSc dissertation, page 37

The distribution of the residuals of D-limonene including and between 18 and 25 days after infestation is represented with a box plot (Figure 5) where the median of the infested fruit is shown to be situated close to the median of the healthy fruit. Consequently, the difference in D-limonene concentration released by healthy and False codling moth infested Cara cara Navel oranges between 18 to 25 days is not significant (p=0.118) (Table 5).

(µg/ml) 6

Concentration of D-limonene

Median 25%-75% -2 Non-Outlier Range Healthy fruit Infested Fruit Outliers Treatment Extremes

Figure 5: Box plot showing the median concentration of D-limonene released from the healthy and infested Cara cara Navel oranges for the experimental period between 18 and 25 days after infestation

MSc dissertation, page 38 The distribution of the 3,7-dimethyl-1,3,6-octatriene residuals and the median for the healthy and infested Cara cara Navel oranges including and between 18 and 25 days after infestation is represented with a box plot (Figure 6). The infested fruit median with a value of 0.067 µg/ml is greater than the median value of 0.038 µg/ml for the healthy fruit (Table 5). Between 18 and 25 days after infestation the concentration of 3,7-dimethyl-1,3,6-octatriene released by False codling moth infested Cara cara Navel oranges was significantly greater than that emitted by the healthy fruit (p=0.049).

0.300

0.250

0.200

0.150

0.100

0.050

0.000

Concentration of 3,7-dimethyl-1,3,6-octatriene (µg/ml) of 3,7-dimethyl-1,3,6-octatriene Concentration Median 25%-75% -0.050 Non-Outlier Range Healthy fruit Infested Fruit Outliers Treatment Extremes

Figure 6: Box plot showing the median concentration of 3,7-dimethyl-1,3,6-octatriene released from the healthy and infested Cara cara Navel oranges for the experimental period between 18 and 25 days after infestation

MSc dissertation, page 39 The response of (E)-4,8-dimethyl-1,3,7-nonatriene to False codling moth infested and healthy Cara cara Navel oranges between 18 and 25 days after infestation is represented with a box plot (Figure 7). The amount of outliers for the residuals of the (E)-4,8-dimethyl-1,3,7-nonatriene infested fruit and the extreme outliers for both treatments can explain the reason for a seemingly lower amount of (E)-4,8-dimethyl- 1,3,7-nonatriene emitted from the infested fruit in comparison to the healthy fruit. The medians of the healthy and infested fruit are similar. It follows that there is no significant difference in the amount of (E)-4,8-dimethyl-1,3,7-nonatriene released by healthy and False codling moth infested Cara cara Navel oranges between 18 and 25 days after infestation (p=0.296) (Table 5).

9000000

8000000 Median 25%-75% Non-Outlier Range 7000000 Outliers Extremes

6000000

5000000

4000000

3000000

2000000

1000000

Concentration of (E)-4,8-dimethyl-1,3,7-nonatriene (Peak area) (E)-4,8-dimethyl-1,3,7-nonatriene of Concentration 0

-1000000 Healthy fruit Infested Fruit Treatment

Figure 7: Box plot showing the median peak area of (E)-4,8-dimethyl-1,3,7- nonatriene released from the healthy and infested Cara cara Navel oranges for the experimental period between 18 and 25 days after infestation

MSc dissertation, page 40 After 18 days of infestation, the distribution of the residuals of caryophyllene between the healthy and infested fruit appeared to show some differences from one another. The distribution of the caryophyllene residuals and the median for the healthy and infested Cara cara Navel oranges including and between 18 and 25 days after infestation is represented with a box plot (Figure 8). The outliers and extreme outlier for the healthy fruit can explain why the amount of caryophyllene appears to be greater for the healthy fruit in comparison to the infested fruit. The medians of the healthy and infested fruit are similar and as a result there is no difference in the amount of caryophyllene released between these points (Table 5). The amount of caryophyllene emitted by healthy and False codling moth infested Cara cara Navel oranges does not differ significantly (p=0.714).

7000000

6000000

5000000

Peak Area) Peak 4000000

3000000

2000000

1000000

Concentration of caryophyllene ( caryophyllene of Concentration

0 Median 25%-75% -1000000 Non-Outlier Range Healthy fruit Infested Fruit Outliers Treatment Extremes Figure 8: Box plot showing the median peak area of caryophyllene released from the healthy and infested Cara cara Navel oranges for the experimental period between 18 and 25 days after infestation

MSc dissertation, page 41 The naphthalene response to False codling moth infested and healthy Cara cara Navel oranges between 18 and 25 days after infestation is represented with a box plot (Figure 9). Although it appears that the healthy fruit released a higher concentration of naphthalene, it is clear that the median of the infested fruit (0.264µg/ml) is quite similar to the median of the healthy fruit (0.272µg/ml) (Table 5). Subsequently the concentration of naphthalene emitted by healthy Cara cara Navel oranges does not differ significantly from the False codling moth infested fruit (p=0.279).

1.000

0.800

0.600

(µg/ml)

0.400

0.200

0.000

Concentration of naphthalene of naphthalene Concentration -0.200

-0.400 Median 25%-75% Healthy fruit Infested Fruit Non-Outlier Range Treatment Outliers

Figure 9: Box plot showing the median concentration of naphthalene released from the healthy and infested Cara cara Navel oranges for the experimental period between 18 and 25 days after infestation

MSc dissertation, page 42 4.2.2. Forcibly infested late harvest Lane late Navel oranges

Initial analysis using scatter plots indicated that the only period where there was a possibility of a statistical difference between the concentrations of the volatile compounds of interest between their respective healthy and infested fruit was after 18 days of infestation. The median values, number of replicates and Mann-Whitney statistics are shown in Table 6 for each of the volatile compounds of interest.

Table 6: Median and Mann-Whitney test values for the response of D-limonene, 3,7- dimethyl-1,3,6-octatriene, (E)-4,8-dimethyl-1,3,7-nonatriene, caryophyllene and naphthalene in healthy and infested Lane late Navel oranges for the experimental period between 18 and 25 days after infestation

Median Median concentration$ concentration$ Mann Whitney Volatile compound N p-value healthy fruit infested fruit Test statistic Z D-limonene 14 1.700 1.515 0.345 0.730 3,7-dimethyl-1,3,6- 0.531 0.596 octatriene 14 0.691 0.125 (E)-4,8-dimethyl- 2.415 0.016 1,3,7-nonatriene 14 11866235 5167761 caryophyllene 14 109460702 71037027 1.794 0.073 naphthalene 14 0.203 0.238 -0.712 0.476

$; Concentrations for D-limonene, 3,7-dimethyl-1,3,6-octatriene, and naphthalene given in µg/ml; amounts for (E)-4,8-dimethyl-1,3,7-nonatriene and caryophyllene given as peak areas.

The D-limonene released by the Lane late Navel oranges 18 and 25 days after infestation is represented with a box plot showing the medians of each treatment (Figure 10). The medians of the healthy and infested fruit are shown to be similar

MSc dissertation, page 43 and as a result there is no difference in the concentration of naphthalene released by these two treatments (Table 6). The difference in D-limonene concentration released by healthy and False codling moth infested Cara cara Navel oranges between 18 to 25 days is not significant (p=0.730).

(µg/ml) 8

Concentration of D-limonene of Concentration 2

0 Median 25%-75% -2 Non-Outlier Range Healthy fruit Infested fruit Outliers Treatment Extremes

Figure 10: Box plot showing the median concentration of D-limonene released from the healthy and infested Lane late Navel oranges for the experimental period between 18 and 25 days after infestation

The distribution of the 3,7-dimethyl-1,3,6-octatriene residuals and the median for the healthy and infested Lane late Navel oranges including and between 18 and 25 days after infestation is represented with a box plot (Figure 11). Although the concentration of 3,7-dimethyl-1,3,6-octatriene appears to be greater for the healthy fruit, the median of the infested fruit is situated quite far from the median of the healthy fruit. However, there is no difference between these points (Table 6) which could possibly be due to a high amount of variation within the data. There is no evidence that the concentration of 3,7-dimethyl-1,3,6-octatriene released by healthy

MSc dissertation, page 44 and False codling moth infested Lane late Navel oranges differs significantly between 18 and 25 days after infestation (p=0.596).

2.200

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Concentration of 3,7-dimethyl-1,3,6-octatriene (µg/ml) 0.000 Median 25%-75% -0.200 Non-Outlier Range Healthy fruit Infested fruit Outliers Treatment Extremes

Figure 11: Box plot showing the median concentration of 3,7-dimethyl-1,3,6- octatriene released from the healthy and infested Lane late Navel oranges for the experimental period between 18 and 25 days after infestation

The response of (E)-4,8-dimethyl-1,3,7-nonatriene and plot of the median values for the healthy and infested Lane late Navel oranges including and between 18 and 25 days after infestation is represented with a box plot (Figure 12). The distribution of the residuals of the healthy and infested fruit showed some variation between these treatments only after 18 days. The medians of the healthy and infested fruit are are shown to be statistically different (Table 6). Between 18 and 25 days after infestation, the amount of (E)-4,8-dimethyl-1,3,7-nonatriene released by False codling moth infested Lane late Navel oranges was significantly lower than that emitted by the healthy fruit (p=0.016).

MSc dissertation, page 45 140000000

120000000

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Concentration of (E)-4,8-dimethyl-1,3,7-nonatriene (Peak area) (Peak (E)-4,8-dimethyl-1,3,7-nonatriene of Concentration Median 25%-75% -20000000 Non-Outlier Range Healthy fruit Infested fruit Outliers Treatment Extremes

Figure 12: Box plot showing the median peak area of (E)-4,8-dimethyl-1,3,7- nonatriene released from the healthy and infested Lane late Navel oranges for the experimental period between 18 and 25 days after infestation

The distribution of the caryophyllene residuals and the median for the healthy and infested Lane late Navel oranges including and between 18 and 25 days after infestation is represented with a box plot (Figure 13). After 18 days of infestation, it appears that the infested fruit produced a lower concentration of caryophyllene; however, the median of the infested fruit is situated close to the median of the healthy fruit and consequently the concentration of caryophyllene emitted by healthy and False codling moth infested Lane late Navel oranges does not differ significantly (p=0.073) (Table 6).

MSc dissertation, page 46 260000000

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(Peak (Peak area)

140000000

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Concentration of caryophyllene ofConcentration caryophyllene 40000000

20000000

0 Median 25%-75% -20000000 Non-Outlier Range Healthy fruit Infested fruit Outliers Treatment Extremes

Figure 13: Box plot showing the median peak area of caryophyllene released from the healthy and infested Lane late Navel oranges for the experimental period between 18 and 25 days after infestation

The median for the response of naphthalene in healthy and infested Lane late Navel oranges including and between 18 and 25 days after infestation is represented with a box plot (Figure 14). The healthy and infested fruit emitted very similar concentrations of naphthalene resulting in their medians being very close and consequently the concentration of naphthalene emitted by healthy Lane late Navel oranges does not significantly differ from the False codling moth infested fruit (p=0.476) (Table 6).

MSc dissertation, page 47 0.700

0.600

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(µg/ml) 0.400

0.300

0.200

0.100

Concentration of naphthalene of naphthalene Concentration

0.000 Median 25%-75% -0.100 Non-Outlier Range Healthy fruit Infested fruit Outliers Treatment Extremes

Figure 14: Box plot showing the median concentration of naphthalene released from the healthy and infested Lane late Navel oranges for the experimental period between 18 and 25 days after infestation

MSc dissertation, page 48 4.3. Major volatile compounds detected using the SPME-GC/MS from naturally infested late harvest Lane late Navel and late harvest Midnight Valencia oranges

The naturally infested Lane late Navel oranges did not respond to infestation, producing no statistically significant change in the concentration of D-limonene (Figure 15) in comparison to healthy fruit (p = 0.400). Contrastingly, the naturally infested Midnight Valencia oranges emitted a significantly higher amount of D- limonene in comparison to the healthy Valencia fruit (p = 0.010).

18 b 16

12 g/mL)

µ 10 8 Healthy fruit a 6 Infested fruit 4 a a

Concentration ( Concentration 2 0 Late NAVELS Late VALENCIAS

Cultivar

Figure 15: Effect of False codling moth larvae infestation of Lane late Navel and Midnight Valencia oranges on the concentration (µg/ml) of D-limonene after natural infestation within the orchard. Bars indicate the standard error of the mean. Bars with the same letter do not differ significantly by Mann-Whitney test (p = 0.05)

MSc dissertation, page 49 There was no statistically significant change in the concentration of 3,7-dimethyl- 1,3,6-octatriene in comparison to healthy fruit for the naturally infested Lane late Navel oranges (p = 0.690). The naturally infested Midnight Valencia oranges responded to infestation by producing a significantly higher concentration of 3,7- dimethyl-1,3,6-octatriene (Figure 16) in comparison to healthy fruit (p = 0.016).

0.60 b

0.50

g/mL) 0.40 µ

0.30 Healthy fruit a 0.20 Infested fruit a a

0.10 Concentration ( Concentration

0.00 Late NAVELS Late VALENCIAS

Cultivar

The naturally infested Lane late Navel oranges showed a significant decrease from the healthy fruit in the concentration of (E)-4,8-dimethyl-1,3,7-nonatriene in response to infestation (p = 0.031) (Figure 17). However, the naturally infested Midnight

MSc dissertation, page 50 Valencia oranges emitted a significantly greater amount of (E)-4,8-dimethyl-1,3,7- nonatriene in comparison to the healthy fruit (p = 0.013).

0.30 a

0.25 a

0.20 g/mL) µ a 0.15 Healthy fruit Infested fruit 0.10 b

0.05 Concentration ( Concentration

0.00 Late NAVELS Late VALENCIAS Cultivar

Neither the naturally infested Lane late Navel oranges or naturally infested Midnight Valencia oranges showed any significant difference from the healthy fruit in the concentration of caryophyllene in response to infestation (p = 0.358; p = 0.245, respectively) (Figure 18).

MSc dissertation, page 51 0.9 a 0.8

0.7 a

0.6 g/mL) µ 0.5 Healthy fruit 0.4 Infested fruit 0.3 b 0.2 b

Concentration ( Concentration 0.1 0.0 Late NAVELS Late VALENCIAS Cultivar

Figure 18: Effect of False codling moth larvae infestation of Lane late Navel and Midnight Valencia oranges on the concentration (µg/ml) of caryophyllene after natural infestation within the orchard. Bars indicate the standard error of the mean. Bars with the same letter do not differ significantly by Mann-Whitney test (p = 0.05)

The naturally infested Lane late Navel and Midnight Valencia oranges showed a significant increase in the concentration of naphthalene in comparison to the healthy fruit (p = 0.036; p = 0.004, respectively) (Figure 19).

MSc dissertation, page 52 1.6 b

1.4

1.2

g/mL) 1.0 µ 0.8 Healthy fruit a a 0.6 Infested fruit 0.4 c

0.2 Concentration ( Concentration 0.0 Late NAVELS Late VALENCIAS

Cultivar

Figure 19: Effect of False codling moth larvae infestation of Lane late Navel and Midnight Valencia oranges on the concentration (µg/ml) of naphthalene after natural infestation within the orchard. Bars with the same letter do not differ significantly by Mann-Whitney test (p = 0.05)

4.4. Comparison of major volatile compounds emitted from naturally infested Midnight Valencia oranges using SPME-GCMS and SEP procedures

It can clearly be seen that the SEP procedure detects a greater concentration of D- limonene than the SPME technique (Figure 20 A) both for the control (p = 0.004) and experiment (p = 0.001). However, there was no significant change in concentration of D-limonene between the control and experiment for both the SPME and SEP techniques (p = 0.966; p = 0.191). The SEP procedure also detects a greater concentration of 3,7-dimethyl-1,3,6-octatriene than the SPME technique (Figure 20 B), both for the control (p = 0.003) and experiment (p = 0.006). A significant change in the concentration of 3,7-dimethyl-1,3,6-octatriene between the control and

MSc dissertation, page 53 experiment using both the SPME technique (p = 0.025) and the SEP technique (p = 0.020) is evident. For (E)-4,8-dimethyl-1,3,7-nonatriene the SEP procedure detects a greater concentration than the SPME technique (Figure 20 C), both for the control (p = 0.001) and experiment (p = 0.003). However, there was no significant change in concentration of (E)-4,8-dimethyl-1,3,7-nonatriene between the control and experiment for both the SPME and SEP techniques (p = 0.134; p = 0.174). For caryophyllene the SEP procedure detects a greater concentration than the SPME technique (Figure 20 D), both for the control (p = 0.042) and experiment (p = 0.048). However there was no significant change in concentration of caryophyllene between the control and experiment for both the SPME and SEP techniques (p = 0.239; p = 0.179). A greater concentration of naphthalene is detected by the SEP procedure than the SPME technique (Figure 20 E) both for the control (p = 0.001) and experiment (p = 0.046). A significant change in the concentration of naphthalene between the control and experiment using both the SPME technique (p = 0.025) and the SEP technique (p = 0.020) is evident. Although the SEP technique shows a higher concentration of naphthalene, the SPME technique shows the same difference between the control and experiment. SPME has a lower degree of sensitivity than SEP, which was able to detect 96% more limonene, 50% more 3,7- dimethyl-1,3,6-octatriene , 59% more (E)-4,8-dimethyl-1,3,7-nonatriene, 50% more caryophyllene and 78% more naphthalene than SPME for the same experiment samples.

MSc dissertation, page 54 A

MSc dissertation, page 55 D

MSc dissertation, page 56 Chapter 5

5. Discussion

The use of an SPME-GC/MS procedure allowed for absorption and detection of volatile compounds from the headspace surrounding intact Navel and Valencia oranges (Citrus sinensis L. Osbeck), both non-infested and infested with false codling moth larvae. The SPME-GC/MS procedure resulted in many different component peaks from each sample. However, several of these volatile compounds repeated with each analysis of each of the different experiments. These particular compounds were noted as the major volatile compounds found by SPME-GC/MS on oranges. These major volatile compounds include D-limonene, 3,7-dimethyl-1,3,6- octatriene, (E)-4,8-dimethyl-1,3,7-nonatriene, caryophyllene and naphthalene. Additional common volatile compounds which were present in some but not all samples are shown in Table 2. Hexyl butanoate is a volatile compound which shows potential to be indicative of False codling moth infestation. Hexyl butanoate is a fruit ester and is commonly found as a component of apple, pears and passion fruit aroma profiles (Kendra et al., 2011). However, in this study this volatile compound was found to have low concentrations released from control fruit but increased concentrations in the naturally infested late Navels and late Valencia fruit, as well as the force infested early and late Navel fruit. Similarly to ethanol, hexyl butanoate is known to increase the ripening of fruit through an ethylene mediated response (Kendra et al., 2011). It is highly possible that hexyl butanoate is indicative of infestation within the fruit as False codling moth infestation is known to cause premature ripening in oranges. Nevertheless this volatile is not an ideal signature compound for infestation as it is not detected in all samples analyzed and is present in a very low concentration range.

Monoterpenes are the most predominantly found volatile compounds surrounding wounded citrus fruit according to Droby et al. (2008). Therefore it is not surprising that limonene was one of the most abundant volatile compounds released by the infested citrus fruit. In response to infestation there was no significant increase in the concentration of limonene in comparison to the control fruit. In both the Cara cara

MSc dissertation, page 57 Navel and Lane Late Navel oranges, the D-limonene concentration increased with time throughout the 25 day experimental period for both the healthy and infested fruit but not in comparison to the respective controls (Figure 5, Figure 10). The concentration of limonene in the naturally infested Lane late Navel oranges had no significant response with infestation (Figure 15). However the naturally infested Midnight Valencia oranges (Figure 15) showed a significant increase in the concentration of limonene released after infestation. When comparing the forcibly infested Lane late Navels and the naturally infested Navels it is evident that there is no difference in the response to infestation, therefore forcibly infesting fruit on the tree is a good representation of the response of D-limonene. After regression analysis of Lane late Navels, an assumption can be made that the lack of response in terms of D-limonene can be owing to the natural increase occurring in both the healthy and naturally infested fruit. It is possible that for Midnight Valencia oranges the increased concentration of D-limonene with infestation is due to the infestation itself and not due to fluctuations by both healthy and infested fruit. The increase in concentration of D-limonene in both the healthy and infested fruit with time is indicative of an increase in this volatile due to ripening. Limonene is known to show a large increase in its release as citrus fruit ripens (Flamini et al., 2010). Although limonene was observed to be an abundant volatile released by these oranges and citrus in general, limonene cannot be regarded as a potential indicator volatile compound for False codling moth infestation in Cara cara Navel and Lane Late Navel oranges. Regression analysis will need to be applied to Midnight Valencia oranges, and any other False codling moth susceptible cultivar, to determine whether the increase in D-limonene concentration is due to infestation. In the event that Valencia cultivars do have a change in D-limonene concentration emitted as a result of False codling moth infestation caution must be made to ensure that if these changes are detected, they are compared to control fruit at the same level of maturity. This will ensure that the changes in limonene are in fact due to larvae penetrating into the fruit and not as a result of ripening.

It was observed that there was no natural fluctuation in the average change in 3,7- dimethyl-1,3,6-octatriene concentration over the 25 day experimental period for both the Cara cara and Lane late Navel oranges. The differences in the medians of the

MSc dissertation, page 58 healthy and control fruit were analyzed after 18 days, where the greatest difference in volatile response was observed. There is some evidence that the response of infested fruit differs significantly from control fruit between 18-25 days after infestation (Figure 6). The comparison of median concentration values (Table 5) indicates that the infested fruit released more 3,7-dimethyl-1,3,6-octatriene than the healthy fruit emitted, with concentrations of 0.067µg/ml and 0.038µg/ml, respectively. There was no response to infestation with respect to the emission of 3,7-dimethyl- 1,3,6-octatriene by the Lane late Navel oranges (Figure 11). Although the median concentration (Table 6) of the infested and healthy fruit do not differ significantly, the healthy fruit emitted 0.691µg/ml in comparison to the lower 0.125µg/ml released by the infested fruit. This coupled to the amount of outliers shown for the infested fruit (Figure 11) indicates that there is a large amount of variation in the data set. In addition the naturally infested Lane late Navel oranges did not change with infestation, however, naturally infested Midnight Valencia oranges (Figure 16) showed a significant increase in the concentration of 3,7-dimethyl-1,3,6-octatriene with infestation. As seen in the case of D-limonene, when comparing the concentration of 3,7-dimethyl-1,3,6-octatriene in the forcibly infested Lane late Navels and the naturally infested Navels it is evident that there is no difference in the response to infestation, therefore forcibly infesting fruit on the tree is a good representation of the response of 3,7-dimethyl-1,3,6-octatriene. As no regression analysis was done on Midnight Valencia oranges, it is possible that the increased concentration of 3,7-dimethyl-1,3,6-octatriene with infestation is due to the infestation itself and not due to fluctuations by both healthy and infested fruit. The increase in 3,7-dimethyl-1,3,6-octatriene emitted from the Midnight Valencia orange samples in response to infestation by False codling moth can be attributed specifically to citrus peel damage caused by penetration of the False codling moth larvae into the fruit (Attaway et al. 1967). Due to the lack of 3,7-dimethyl-1,3,6- octatriene produced in both the early and late harvested Navel oranges, it may not be possible that this volatile compound be used as a signature compound for infestation in all cultivars. However, if it were combined with other compounds in a volatile profile of compounds indicative of infestation, this volatile bouquet could serve a good purpose in particular cultivars. D-limonene and 3,7-dimethyl-1,3,6- octatriene (also known as trans-β-ocimene) have been found in several other studies related to herbivore induced fruit volatiles (Hern and Dorn 2002; Birla et al. 2005;

MSc dissertation, page 59 Kendra et al. 2011). These volatile compounds are not thought to be produced by the infestation of False codling moth or by the insect itself but rather caused by the damage to the fruit peel. According to Attaway et al. (1967) terpenes D-limonene and 3,7-dimethyl-1,3,6-octatriene make up a total of 93% and 2.7% of the composition of citrus peel oils.

There was no response to infestation with respect to the emission of (E)-4,8- dimethyl-1,3,7-nonatriene by the Cara cara Navel oranges which is shown by the Mann-Whitney statistical test (Table 5). Although the median concentration (Table 5) of the infested and healthy fruit do not differ significantly, the healthy fruit emitted a larger amount of (E)-4,8-dimethyl-1,3,7-nonatriene in comparison to the healthy fruit. This coupled to the number of outliers shown for the infested fruit (Figure 7) indicates that there is a large amount of variation in the data set. However, the Lane late Navel oranges responded to infestation by False codling moth by having a significantly lower amount of (E)-4,8-dimethyl-1,3,7-nonatriene released (Figure 12). A similar response by the forcibly infested Lane late Navel oranges to False codling moth was observed by the naturally infested oranges which indicated a significant decrease with infestation in contrast to the naturally infested Midnight Valencia oranges (Figure 17) which released a significantly higher concentration of (E)-4,8-dimethyl- 1,3,7-nonatriene in comparison to control fruit. The difference in response of the fruit sample to the infestation by Cara cara Navel oranges and Lane late Navel oranges in comparison to the naturally infested Midnight Valencia samples could be due to the colder temperatures experienced in the orchards during the late harvest season. This would have resulted in the repression of the hatching of the False codling moth eggs or growth of the larvae which have optimum growth at 27°C. (E)-4,8-dimethyl- 1,3,7-nonatriene has been shown to be used by several insects in insect chemical communication systems including that of Lepidoptera (Knight et al., 2011). The early larval development response of (E)-4,8-dimethyl-1,3,7-nonatriene can be attributed to the fact that this volatile compound is produced as a plant’s indirect defence. It is known that several terpenes such as (E)-4,8-dimethyl-1,3,7-nonatriene are produced and released by several plant species following insect herbivore damage as a part of the particular plant’s indirect defence (Ruther and Fürstenau., 2005; Turlings and Tumlinson, 1992). This indirect defence results in plants releasing volatile

MSc dissertation, page 60 compounds in an attempt to attract predators of the primary herbivores and help the secondary herbivores locate their prey to prevent damage to the host plant (Hern and Dorn, 2001). Therefore, the release of (E)-4,8-dimethyl-1,3,7-nonatriene from the fruit is a result of the plant’s indirect defence to herbivore damage caused by False codling moth larvae. This can give reason to the immediate increase in (E)- 4,8-dimethyl-1,3,7-nonatriene in the forcibly infested Lane late Navel oranges as this compound may be released in response to the initial penetration of the larvae, as well as to the subsequent decline in concentration as the larvae is within the fruit and no further damage is occurring to the plant. (E)-4,8-dimethyl-1,3,7-nonatriene has potential to be used as a signature compound in particular cultivars indicating False codling moth infestation on these citrus cultivars in combination with other signature compounds which are indicative of early infestation. A collection of volatiles which respond much like (E)-4,8-dimethyl-1,3,7-nonatriene would allow compounds that are not sufficiently reliable or reproducible in all interested cultivars to become part of a reliable and indicative bouquet.

There was no statistically significant difference between the concentration of caryophyllene emitted by the healthy and infested fruit for both the Cara cara Navel oranges and Lane late Navel oranges (Tables 5 & 6). Both the healthy and infested fruit data sets had a large amount of variation in the caryophyllene values, with both data sets showing outliers, which influenced the outcome of the comparison of these two data sets (Figure 8; Figure 13). In addition, caryophyllene was not able to show any response to infestation of False codling moth in the naturally infested Lane late Navel and Midnight Valencia oranges (Figure 18). Therefore it can be stated that caryophyllene cannot serve as an indicator volatile compound for the infestation of False codling moth in the cultivars analysed.

The forcibly infested Cara cara Navel oranges and Lane late Navel oranges did not produce significantly greater amounts of naphthalene in response to False codling moth infestation (Tables 5 & 6). Although the median concentration naphthalene from the infested Lane late Navel oranges was higher than that from healthy fruit (Figure 14), this difference was not shown to be significant. It is shown that the

MSc dissertation, page 61 healthy fruit contains outliers caused by variation in the data which are clearly ensuring no difference is indicated between the healthy and infested fruit. Initial regression analysis showed that naphthalene from the fruit infested with False codling moth significantly increased, however this did not get to a point where the infested fruit differed significantly from the healthy controls and therefore the medians of the fruit after 18 days were analyzed. The concentration of naphthalene in the naturally infested Lane late Navel and Midnight Valencia oranges (Figure 19) was significantly higher than the healthy fruit. The majority of the fruit from both the naturally infested samples were in the second or third instar within the fruit. At this stage of development visual signs of infestation are starting to occur in the form of discolouration of the entire fruit and yellow discoloured spots surrounding the area of penetration by the larvae. This concurs with the response of the forcibly infested Lane late Navels where a greater difference between the infested and healthy fruit was seen with time, which is directly related to the development of the larvae. Naphthalene is possibly produced due to larval feeding and development within the fruit as concentrations remained higher in infested fruit than in the control fruit throughout the experimental period and even increased with time. Naphthalene showed great reproducibility and consistently showed an increased amount of emission with infestation for all cultivars. Naphthalene would be a good indicator of False codling moth infestation, however, not primarily for the detection of early infestation.

Differences in the volatile profile detected from healthy fruit in comparison to previous studies on different citrus fruits and varieties were noted. A similar phenomenon was noted by Hern and Dorn (2002) in comparison to Boeve et al., (1996) which was said to be due to varietal or methodological differences between the studies. This study showed that there were differences in volatile compound concentration in response to infestation in different varieties. However, these differences were small and all responses showed the same change in emission in comparison to healthy fruit. It is evident that there is a large amount of variation in the data sets of several of the volatile compounds. These unknown variables are more than likely changing the outcome and statistical significance of the findings.

MSc dissertation, page 62 These confounding variables could possibly include the weather changes in the orchard, the conditions during harvesting and infestation of fruit, the time of day and amount of time before analysis or simply the manner in which analysis is performed needs to be more consistent. These will need to be considered and possibly controlled in further studies in order to lessen the amount of variability in the data. A comparable difference in concentration of volatiles was shown for the naturally infested Lane late Navel oranges and naturally infested Midnight Valencia oranges. According to Boeve et al., (1996) a major factor in the emission of volatiles from fruit is the time of year of the harvest. This shows that in order for a volatile detection apparatus for early infestation to be developed, all varieties of fruit that are susceptible to False codling moth infestation will need to be assessed.

The comparison of the SPME and SEP techniques shows a greater amount of each of the major volatile compounds are concentrated on the SEP’s absorptive material (Figure 20 A-E). This resulted in a significantly higher amount of D-limonene, 3,7- dimethyl-1,3,6-octatriene, (E)-4,8-dimethyl-1,3,7-nonatriene and naphthalene detected using the SEP technique over the SPME technique. No significant change in volatile compound was recorded for D-limonene, (E)-4,8-dimethyl-1,3,7-nonatriene and caryophyllene after infestation for both the SPME and SEP analysis, possibly due to the limited replicates of these samples (n = 6). However, significant increases in the amount of 3,7-dimethyl-1,3,6-octatriene and naphthalene were recorded for both the SPME and SEP analysis.

Although the SEP analysis showed a higher amount of both 3,7-dimethyl-1,3,6- octatriene and naphthalene, the SPME technique shows a similar relative change between the control and experiment as well as a similar significant difference in each case. The SEP did not show any additional compounds to the common compounds found using the SPME technique. This preliminary study shows that SEP is far more sensitive with far more adsorption and abundance of the compounds of interest than the SPME procedure. That being said, the SPME is able to detect the same differences between control and infested fruit, although at a lower amount to the SEP. A disadvantage of the SEP technique for application on citrus fruit response to infestation is the duration of adsorption of 2-24 hours, whereas the SPME adsorption

MSc dissertation, page 63 requires only 10 minutes. SEP, however, provides the advantage of storage of adsorption fibres for manual injection at a later time. Another advantage of the SEP is the low cost of the fibres, and the absence of an adapter means that more than one replicate of the same sample can be done at the same time reducing the time required. Implementation of the SEP technique on certain gas chromatographs requires small inexpensive changes to the injector hardware, whereas other more sophisticated systems require the replacement of the septum cap and the septum supporting hardware (Burger et al., 2006). Additional work using SEP with a larger amount of replicates will need to be done in order to determine whether this more sensitive technique can possibly detect if there are any trace volatile compounds present in infested fruit and not in healthy fruit.

5.1. Conclusions

This study showed that volatile compounds diagnostic of Thaumatotibia leucotreta infestation of orange fruit are possible. The major volatile compounds which were reproducible in each sample include D-limonene, 3,7-dimethyl-1,3,6-octatriene, (E)- 4,8-dimethyl-1,3,7-nonatriene, caryophyllene and naphthalene. The volatile compounds that have potential to be proven as an indicator of infestation include, hexyl butanoate, (E)-4,8-dimethyl-1,3,7-nonatriene, and naphthalene.

Although limonene was observed to be one of the most abundant volatile compounds released, it cannot be utilized as an indicator of False codling moth infestation in Cara cara Navel and Lane Late Navel oranges, further investigation will tell if this is the case for all citrus cultivars susceptible to this pest. The release of (E)- 4,8-dimethyl-1,3,7-nonatriene immediately after infestation can be attributed to the fact that this volatile compound is typically produced as a plant’s indirect defense and is highly possible that this is the case with these cultivars. Naphthalene alone is the best chance of detecting False codling moth infested fruit from all these particular cultivars; however, this volatile shows its greatest variation from non-infested fruit

MSc dissertation, page 64 late in the development stage where visual signs are already apparent. It would be suggested that a bouquet of volatile compounds could be exploited using signature compounds that may differ with early or late harvest or cultivar.

Compounds produced that were indicative of infestation were not insect produced but naturally produced fruit volatiles emitted at higher levels, as a result of the insect within the fruit. Volatile compounds were produced due to damage to the fruit’s peel or pulp through penetration of the larvae. This was due either to damage to the pulp through larval development and feeding within the fruit or because the fruit responded to the infestation by an indirect chemical response to attract secondary predators. These major volatile compounds, which were reproducible in each sample, could together potentially provide a practical signature volatile profile (bouquet) for False codling moth infestation of orange fruit for these varieties.

A definite requirement is the need for reproducibility of the procedure and further concentration of volatile compounds in order to decrease the error between samples enabling the detected trends to be statistically relevant. This could be achieved by the higher concentrations and higher sensitivity achieved using the SEP technique which needs to be coupled to good reproducibility and optimization of the procedure.

If this volatile profile is consistently detected by further infested fruits and the trends are revealed to be indicative of infestation, an indicator profile for infested fruit can be exploited for the development of a rapid and reliable screening technique for early detection of False codling moth infestation in oranges post harvest.

5.2. Future work

 Volatile emissions of fruit infested while attached to the tree and detached infested fruit should be compared to healthy fruit of the same age and picked

MSc dissertation, page 65 at the same time. This will determine whether further studies need to continue on fruit in the orchard or whether lab studies can accurately depict volatile changes. If the latter is possible, it will make the study easier and more reliable, due to eliminating other variables present in the field allowing more replicates to be analyzed.

 Volatiles from different citrus cultivars and varieties should be trapped using SEP and detected using GC-MS. Several replicates could be performed in pairs (healthy/infested fruit) as a function of time from infestation. Once again various data analysis techniques could then be applied to the data so as to estimate: (i) differences in types of volatiles between pairs; (ii) differences in the relative amounts of volatiles between pairs; and (iii) variation in volatile profile/amounts as a function of time since infestation (larval development).

 False codling moth adults should be subjected to Y-tube olfactometer tests to establish (i) whether female False codling moth adults are attracted to particular volatiles released by citrus fruit, (ii) whether certain citrus varieties or cultivars have higher levels of this volatile and (iii) if the occurrence of this volatile can be correlated to that of False codling moth susceptible citrus cultivars and varieties.

 The most feasible rapid volatile screening technique, in terms of sensitivity, efficiency and affordability should be determined. Detection of volatiles using a variety of rapid volatile screening techniques (Ultra fast GC, zNose technology) should be correlated to the volatile profiles detected by the SEP paired with GC-MS. Any prominent differences between type and/or amount of volatile, or even complete volatile profiles by this rapid screening technique, can be studied further with a view to identification of infested fruit (possibly in a blind study using force infested fruit).

 Once volatiles indicative of False codling moth infestation and False codling moth susceptibility in particular citrus cultivars are determined, gene

MSc dissertation, page 66 expression profiling could be done using qPCR in order to determine whether there are changes in enzyme expression, as well as activity. Knowing the changes in enzyme activity will bring us closer to understanding what metabolic activities occur in fruit with False codling moth susceptibility or False codling moth infestation, identification of the origin of these volatiles and an understanding of how these volatile profiles can be used to our advantage in False codling moth control and detection.

 The detection of larvae by direct measurement of the genetic material through rDNA analysis or through serological methods such as larval antigen detection can be used to compare with volatile methods.

 This study has the potential for future research in the determination of volatiles indicating internal diseases (e.g. Alternaria), external diseases (e.g. citrus black spot, Guignardia citricarpa), differences in fruit acidity and sugar levels or natural internal physical and physiological disorders (e.g. rind creasing, granuation) in fruit.

MSc dissertation, page 67 Chapter 6

6. Appendix

100000000

90000000 y = 9E+07x - 4E+06 80000000 R² = 0.9883

70000000

60000000

50000000 Peak Area Peak 40000000

30000000

20000000

10000000

0 0 0.2 0.4 0.6 0.8 1 1.2 Concentration (µglmL)

Figure 21: Limonene standard concentrations and their respective peak areas used for quantification

MSc dissertation, page 68 4E+09

y = 2E+10x + 1E+09 3.5E+09 R² = 0.8852

3E+09

2.5E+09

2E+09 Peak Area Peak 1.5E+09

1E+09

500000000

0 0 0.02 0.04 0.06 0.08 0.1 0.12 Concentration (µg/mL)

Figure 22: 3,7-dimethyl-1,3,6-Octatriene standard concentrations and their respective peak areas used for quantification

4.5E+09

4E+09

3.5E+09

3E+09

2.5E+09

y = 4E+10x + 3E+08 Peak Area Area Peak 2E+09 R² = 0.9721

1.5E+09

1E+09

500000000

0 0 0.02 0.04 0.06 0.08 0.1 0.12 Concentration (µg/mL)

Figure 23: Naphthalene standard concentrations and their respective peak areas used for quantification

MSc dissertation, page 69

Figure 24: Chromatogram showing the peak height, peak area and retention time for 1 µg/ml of D-Limonene commercial standard used for quantification

MSc dissertation, page 70 A b u n d a n c e

TIC: NAPHTHALENE 3.D\data.ms

3 . 8 e + 0 7 3 . 6 e + 0 7 3 . 4 e + 0 7 3 . 2 e + 0 7 3 e + 0 7 2 . 8 e + 0 7 2 . 6 e + 0 7 2 . 4 e + 0 7 2 . 2 e + 0 7 2 e + 0 7 1 . 8 e + 0 7 1 . 6 e + 0 7 1 . 4 e + 0 7 1 . 2 e + 0 7 1 e + 0 7 8 0 0 0 0 0 0 6 0 0 0 0 0 0 4 0 0 0 0 0 0 2 0 0 0 0 0 0

1 . 0 0 2 . 0 0 3 . 0 0 4 . 0 0 5 . 0 0 6 . 0 0 7 . 0 0 8 . 0 0 9 . 0 0 1 0 . 0 0 T im e - - >

Figure 25: Chromatogram showing the peak height, peak area and retention time for 1 µg/ml of 3,7-dimethyl-1,3,6-Octatriene commercial standard used for quantification

MSc dissertation, page 71 A b u n d a n c e

TIC: TERPINENE 1.D\data.ms

4 5 0 0 0 0 0

4 0 0 0 0 0 0

3 5 0 0 0 0 0

3 0 0 0 0 0 0

2 5 0 0 0 0 0

2 0 0 0 0 0 0

1 5 0 0 0 0 0

1 0 0 0 0 0 0

5 0 0 0 0 0

1 . 0 0 2 . 0 0 3 . 0 0 4 . 0 0 5 . 0 0 6 . 0 0 7 . 0 0 8 . 0 0 9 . 0 0 1 0 . 0 0 T im e - - >

Figure 26: Chromatogram showing the peak height, peak area and retention time for 1 µg/ml of Naphthalene commercial standard used for quantification

MSc dissertation, page 72 Abundance

TIC: CC NAVEL CONTROL A 5.D\data.ms 7500000

7000000

6500000

6000000

5500000

5000000

4500000

4000000

3500000

3000000

2500000

2000000

1500000

1000000

500000

2.00 4.00 6.00 8.00 10.00 12.00 14.00 Time-->

Figure 27: Typical chromatogram from the healthy fruit samples showing the peak height, peak area and retention time of the volatiles found in the headspace of intact oranges

MSc dissertation, page 73 Abundance

TIC: CC NAVEL 2ND SAMPLE 7.D\data.ms 1.4e+07 1.3e+07 1.2e+07 1.1e+07 1e+07 9000000 8000000 7000000 6000000 5000000 4000000 3000000 2000000 1000000

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 Time-->

Figure 28: Typical chromatogram from the False codling moth infested fruit samples showing the peak height, peak area and retention time of the volatiles found in the headspace of intact oranges

MSc dissertation, page 74 Chapter 7

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