INTERACTIONS BETWEEN BRASSICACEOUS PLANTS AND THE EFFECTS ON THE PLANTS’ SUSCEPTIBILITY TO
OVIPOSITION BY PEST LEPIDOPTERA IN AN OZONE ENRICHED ENVIRONMENT
Gaukhar Malikova MSc Thesis Green Biotechnology and Food Security University of Eastern Finland Faculty of Science and Forestry Department of Environmental and Biological Sciences September 2, 2016
UNIVERSITY OF EASTERN FINLAND
Faculty of Science and Forestry, Department of Environmental and Biological Sciences Green Biotechnology and Food Security program
Gaukhar Malikova: Interactions between brassicaceous plants and the effects on the plants’
susceptibility to oviposition by pest Lepidoptera in an ozone enriched environment Master’s thesis, 47 pages
Supervisors: James D. Blande, Ph.D., Academy Research Fellow; P. Sarai Girón-Calva, MSc, Researcher
September 2, 2016
Keywords: Volatile organic compounds (VOCs), plant-plant signalling, tropospheric ozone, cabbage plants, Pieris brassicae, Plutella xylostella
ABSTRACT
Herbivore-damaged plants emit a blend of volatile organic compounds (VOCs), known as herbivore-induced plant volatiles (HIPVs). By emitting these compounds, plants can signal to undamaged neighbouring plants, to increase their resistance to herbivores. These mechanisms help plants to protect plants from biotic stress. In addition, HIPVs play an important role in plant-plant signalling. Tropospheric ozone is the major air pollutant that reduces VOC- mediated signalling by degrading many HIPVs. Negatively influence to the agriculture and natural ecosystems, depends on concentration of ozone.
The main aim of this research was to test if intact cabbage plants exposed to HIPVs from conspecific and/or heterospecific neighbouring plants are less susceptible to oviposition by the pest Lepidopteran Plutella xylostella or not. In addition, the research aimed to assess whether increased ozone levels eliminate or reduce the plant-plant signalling. In this research, the model system included cabbage and broccoli as HIPV emitting plants, and cabbage as HIPV receiving plants. Also, third-instar Pieris brassicae larvae were used as a plant damaging herbivore, and 4-5 day old Plutella xylostella adults were used in oviposition experiments. Experiments were conducted in the laboratory and field conditions, under ambient ozone and elevated ozone concentrations and both comprised two phases: (1) exposure of undamaged plants to HIPVs from an herbivore-damaged neighbours and (2) an oviposition preference test with plants exposed to differently induced neighbours.
Plants exposed to HIPVs from cabbage plants damaged by P. brassicae were less susceptible of cabbage receiver plants to oviposition by P. xylostella, than plants exposed to undamaged plants, under ambient ozone conditions. However, this effect was impaired under elevated ozone (80ppb) conditions in the field. The findings indicate that ozone could significantly disturb the volatile mediated plant-plant signalling.
УНИВЕРСИТЕТ ВОСТОЧНОЙ ФИНЛЯНДИИ
Факультет естественных наук и лесного хозяйства, Кафедра окружающей среды и биологических наук
Программа Зеленая Биотехнология и Пищевая Безопасность
Гаухар Маликова: Влияние озоно-обогащенной среды на взаимодействия капустных растений и их восприимчивость к откладке яиц чешуекрылых вредителей
Магистерская диссертация, 47 страниц
Руководители: Джеймс Д. Бленд, Ph.D., научный сотрудник Академии; П. Сарая Гирон- Кальва, MSc, исследователь
Сентябрь 2, 2016
Ключевые слова: Летучие органические соединения (ЛОС), коммуникация между растениями, тропосферный озон, капустные растения, Pieris brassicae, Plutella xylostella
АБСТРАКТ
Растения поврежденные от травоядных организмов выделяют смесь летучих органических соединений (ЛОС), известные как травоядно-индуцированные летучие растительные вещества (HIPVs). Растения, выпуская эти соединения могут посылать сигналы к неповрежденным растениям-соседям, повысить их устойчивость к травоядным. Эти механизмы помогают растениям защититься от биотических стрессов.
HIPVs также играют важную роль в коммуникации между растениями. Тропосферный озон является главным загрязнителем воздуха, который уменьшает ЛОС- посредственную коммуникацию, разрушая многих HIPVs. Озон, в зависимости от концентрации имеет негативное влияние на сельское хозяйство и природные экосистемы.
Основная цель этого исследования состояла в том, чтобы оценить восприимчивость к откладке яиц вредителя Plutella xylostella,, которое относится к семейству чешуекрылые, на неповрежденных капустных растениях, подверженных HIPVs от конспецифичных и/или гетероспецифических растений-соседей. Следующей целью было проверить насколько, обогащенные уровнем озона, могут устранять или уменьшать коммуникацию между растениями. В этом исследовании модельными системами являются такие растения, как капуста и брокколи, которые выступают как HIPV излучающие растения, и капуста, как HIPV принимающее растение. А также, личинки травоядных насекомых Pieris brassicae III стадии были использованы в качестве насекомых, которые повреждали растения и 4-5 -дневные взрослые Plutella xylostella были использованы в экспериментах для откладки яиц. Эксперименты проводились в полевых и лабораторных условиях, при атмосферной и повышенной концентрацией озона, где они состояли из двух фаз: (1) воздействие неповрежденных растений на HIPVs сигнал от поврежденного травоядными насекомыми растений- соседей и (2) тест на предпочитаемость откладки яиц растениями, подвергнутыми разными индуцированными растениями-соседей.
Растения, подвергнутые воздействию травоядно-индуцированных летучих растительных веществ (HIPVs) от капустных растений, поврежденные P. brassicae были менее восприимчивы к яйцекладке P. xylostella, чем растения, подвергнутые неповрежденными растениями, при условии атмосферного озона. Тем не менее , этот эффект нарушается при повышенном содержании озона (80ppb) в полевых условиях.
Полученные результаты свидетельствует о том, что озон может существенно нарушить коммуникацию между растениями.
ШЫҒЫС ФИНЛЯНДИЯ УНИВЕРСИТЕТІ
Жаратылыстану ғылымдары және орман шаруашылығы факультеті, Қоршаған орта және биология ғылымдары кафедрасы
Жасыл Биотехнология және Тағам Қауіпсіздігі бағдарламасы
Гаухар Маликова: Озон байытылған ортаның қырыққабат өсімдіктерінің өзара байланысына және қабыршаққанаттылардың жұмыртқа салуына сезімталдық әсері Магистрлік диссертация, 47 бет
Ғылыми жетекшілер: Джеймс Д. Бленд, Ph.D., Академияның ғылыми қызметкері;
П. Сарая Гирон-Кальва, MSc, зерттеуші Қыркүйек 2, 2016
Түйін сөздер: Ұшқыш органикалық қосылыстар, өсімдік аралық байланыс, тропосфералық озон, қырыққабат өсімдіктері, Pieris brassicae, Plutella xylostella
ТҮЙІНДЕМЕ
Шөпқоректі ағзалардан зақымдалған өсімдіктер, өсімдіктердің шөпқоректі- индукцияланған ұшқыш заттары (HIPVs) ретінде белгілі, ұшқыш органикалық қосылыстар (ҰОҚ) қоспасын бөледі. Өсімдіктер осы қосылыстарды бөле отырып, зақымдалмаған көршілес өсімдіктерге, олардың шөпқоректілерге қарсы төзімділігін арттыратын алатын белгі жібереді. Бұл механизмдер өсімдіктерге биотикалық стресстерден қорғану үшін көмектеседі. Сонымен қатар, HIPV өсімдік аралық байланыста маңызды рөл атқарады. Тропосфералық озон көптеген HIPV-ларды жоя отырып, ұшқыш органикалық қосылыстар-аралық байланысты азайтатын, ауаның негізгі ластаушысы болып табылады. Озон, белгілі концентрациясына байланысты ауыл шаруашылығы мен табиғи экожүйелерге кері әсер етеді.
Зерттеудің негізгі мақсаты конспецификалық және/немесе гетероспецификалық көршілес өсімдіктердің HIPVs-на бақыланған, зақымдалмаған қырыққабат өсімдіктерінің қабыршаққанатты Plutella xylostella зиянкесінің жұмыртқа салуына сезімталдығын анықтау болған. Сонымен қатар, зерттеудің мақсаты озонның байытылған деңгейі, өсімдік аралық байланысты қаншалықты азайта немесе жоя алатынын анықтау болды. Бұл зерттеуде модельдік жүйе ретінде қырыққабат және брокколи, HIPV қабылдаушы өсімдіктер, және қырыққабат HIPV шығарушы өсімдік ретінде қарастырылды. Сондай-ақ, III кезеңдік Pieris brassicae дернәсілдері өсімдікті зақымдаушы шөпқоректі жәндіктер ретінде қолданылды, және 4-5 күндік Plutella xylostella ересектері жұмыртқа салу тәжірибелерінде қолданылды. Тәжірибелер озонның атмосфералық және жоғары концентрациясында, далалық және зертханалық жағдайда жүргізілді, сондай-ақ екі фазадан тұрды: (1) зақымдалмаған өсімдіктердің шөпқоректі жәндіктермен зақымдалған көршілес өсімдіктердің HIPVs сигналына әсері, және (2) әртүрлі индукцияланған көршілес өсімдіктермен бақыланған өсімдіктерге зиянкестердің жұмыртқа салу бейімділігін анықтайтын тест.
Атмосфералық озон жағдайында, қырыққабат өсімдіктерінің шөпқоректі- индукцияланған ұшқыш заттардың (HIPVs) әсеріне ұшырап, P. brassicae зиянкесімен зақымдалған өсімдіктер, зақымдалмаған өсімдіктерге қарағанда P. xylostella-ның жұмыртқа салуына сезімталдығы аз болды. Алайда, бұл әсер далалық жағдайда, озонның жоғары деңгейінде (80ppb) бұзылды. Алынған нәтижелер көрсеткендей озон өсімдік аралық байланысты елеулі түрде бұзады.
ACKNOWLEDGEMENT
I want to thank my supervisors Dr. James D. Blande and P. Sarai Girón-Calva, MSc for their guidance and direction during my research. My sincere gratitude goes to James for his professionalism, advice and for helping me move on during the difficult moments in my thesis writing. My deepest gratitude goes to Sarai for guiding me during the internship, helping in data collection and statistical analyses. I am very thankful for her suggestions, giving me right direction and helping me in each stages of this thesis. I am very grateful to my coordinator Roseanna Avento for giving me motivation, encouragement, advice and recommendations throughout this thesis.
I would like to thank all staff of the University of Eastern Finland, especially in the Department of Environmental and Biological Sciences who gave me high knowledge and study skills during the study year. I will never forget interesting and wonderful moments in Kuopio.
In addition, I would like to acknowledge the administration of my home university (Kazakh National Agrarian University) for giving me the opportunity to study in the program Green Biotechnology and Food Security and for the financial support during the academic year in UEF.
Lastly, I want to thank to my family and best friends who supported and believed in me. Their care and support helped me stay focused on my Master’s thesis.
Thank you!
ABBREVIATIONS
AmbCR AmbIR ANOVA C6
CO CH4 DMNT EFN FACE GLV HIPV HR
JA-pathway LED
LSD MeSA NOx
OzoCR OzoIR O3
PCD
P. xylostella P. brassica Ppb ROS UV VOCs
Ambient control receiver treatment Ambient infested receiver treatment Analysis of variation
6 atoms of carbon Carbon monoxide Methane
8-dimethyl-1,3,7-nonatriene Extrafloral nectar
Free air concentration enrichment Green-leaf volatile
Herbivore-induced plant volatile Hypersensitive reactions
The jasmonate signal pathway Light-emitting diode
Least significant difference Methyl salicylate
Nitrogen oxides
Ozone control receiver treatment Ozone infested receiver treatment Ozone
Programmed cell death Plutella xylostella Pieris brassica Parts per billion
Reactive oxygen species Ultraviolet
Volatile organic compounds
CONTENT
ACKNOWLEDGEMENT ... 5
ABBREVIATIONS ... 6
1. INTRODUCTION ... 9
2. LITERATURE REVIEW ... 11
2.1. VOLATILES RELEASE IN RESPONSE BIOTIC AND ABIOTIC STRESSES ... 11
2.1.1. Role of volatiles as direct and indirect defences of plants ... 12
2.1.2. Role of volatiles in plant-plant signalling ... 13
2.1.3 Active and passive plant-plant interactions ... 14
2.2 EFFECT OF OZONE ON PLANTS ... 16
2.2.1 Effects of ozone on VOCs and plant-plant signalling ... 18
2.3 BRASSICA OLERACEA L. VAR. CAPITATА (CABBAGE) AND BRASSICA OLERACEA VAR. ITALICA (BROCCOLI) AS STUDY SYSTEMS FOR FIELD AND LABORATORY EXPERIMENTS ... 19
2.4 LEPIDOPTERA PESTS ... 20
2.4.1 Pieris brassicae ... 20
2.4.2 Plutella xylostella ... 21
3. OBJECTIVES ... 23
4. MATERIALS AND METHODS ... 24
4.1 PLANTS AND INSECTS ... 24
4.2 FREE AIR CONCENTRATION ENRICHMENT (FACE) FACILITIES ... 24
4.3 CONTROLLED ENVIRONMENT CHAMBERS AND OZONE EXPOSURE SYSTEM ... 25
4.4 PLANT-PLANT COMMUNICATION BETWEEN CABBAGE PLANTS UNDER FIELD CONDITIONS AND OVIPOSITION PREFERENCE TEST OF P. XYLOSTELLA ... 26
4.4.1 Phase 1 Exposure to HIPVs ... 26
4.4.2 Phase 2 Oviposition preferences of P. xylostella ... 27
4.5 PLANT-PLANT COMMUNICATION BETWEEN CABBAGE PLANTS UNDER LABORATORY CONDITIONS AND OVIPOSITION PREFERENCE TEST OF P. XYLOSTELLA ... 27
4.5.1 Phase 1 Exposure to HIPVs ... 27
4.5.2 Phase 2 Oviposition preferences of P. xylostella ... 28
4.6. PLANT-PLANT COMMUNICATION BETWEEN BROCCOLI AND CABBAGE PLANTS UNDER LABORATORY CONDITIONS AND OVIPOSITION PREFERENCE TEST OF P. XYLOSTELLA ... 29
4.6.1 Phase 1 Exposure to HIPVs ... 29
4.6.2 Phase 2 Oviposition preferences of P. xylostella ... 29
4.7 DATA ANALYSIS ... 30
5. RESULTS ... 31
5.1 PLANT-PLANT COMMUNICATION BETWEEN CABBAGE PLANTS UNDER FIELD CONDITIONS AND OVIPOSITION PREFERENCE TEST OF P. XYLOSTELLA ... 31
5.2 PLANT-PLANT COMMUNICATION BETWEEN CABBAGE PLANTS UNDER LABORATORY CONDITIONS AND OVIPOSITION PREFERENCE TEST OF P. XYLOSTELLA ... 32
5.3 PLANT-PLANT COMMUNICATION BETWEEN BROCCOLI AND CABBAGE PLANTS UNDER LABORATORY CONDITIONS AND OVIPOSITION PREFERENCE TEST OF P. XYLOSTELLA ... 33
6. DISCUSSION ... 34
7. CONCLUSION ... 37
REFERENCES ... 38
1. INTRODUCTION
All green plants are a food source for herbivores (Zakir et al., 2009). Plants respond to herbivory by emitting a variety of volatile organic compounds (VOCs), known as herbivore- induced plant volatiles (HIPVs) (Campos et al., 2008). During growth, plants produce about 100 000 known chemical products of which, 1700 are volatile (Dicke and Loreto, 2010). Plant VOC emissions are released from a variety of plant vegetative tissues and diffuse into the atmosphere (Kask et al., 2013). HIPVs play roles in direct and indirect defences; direct defences include toxic and herbivore-repellents chemicals, whereas indirect defences include the attraction of natural enemies of herbivores (Arimura et al., 2009). HIPVs also play a role in plant-plant signalling (Frost et al., 2008) and can protect plants from biotic factors such as pathogens and herbivore feeding and abiotic factors such as drought, heat stress and ozone (Holopainen, 2004).
HIPVs are said to play an important role as mediators of plant-plant signalling (Pinto et al., 2010), which is one of the controversial issues in modern ecology. One of the main points of controversy has been the distance at which HIPV signals can be received by a plant (Frost et al., 2008). The distances at which volatiles can affect other plants mainly depends on abiotic factors (Heil and Karban, 2010), for example, field experiments which were done with wild tobacco (Nicotiana attenuata) exposed to clipped sagebrush (Artemisia tridentata), and lima bean (Phaseolus lunatus L.) exposed to conspecifics, showed that communication between plants was only effective over short distances of between 30 cm and 1 m (Heil and Adame- Álvarez, 2010; Karban et al., 2003). Blande et al. (2010) observed that in the presence of ozone, this distance could decrease. This leads to degradation of volatile signals between emitter and receiver plants (Li and Blande, 2015).
Ozone (O3) is recognized as a rural air pollutant whose phytotoxicity and reactivity make it a significant gas in terms of plant-plant signalling (Li and Blande, 2015). Tropospheric ozone has a negative influence on agriculture and natural ecosystems (Shindell et al., 2009). In addition, tropospheric ozone is produced in significant amounts as a result of anthropogenic activities (Ainsworth et al., 2012), with ozone concentrations increasing substantially since industrialization (Sitch et al., 2007). The influence of ozone on a plant depends on the genetics and variety of the plant and on the concentration of ozone (Heck and Miller, 1994).
Ozone reacts with many HIPVs and by degrading them, may reduce VOC-mediated
signalling. Ozone of an 80 ppb mixing ratio significantly decreases the distance at which plant-plant signalling is effective (Blande et al., 2010). In addition, ozone affects the efficiency of feeding by arthropods and orientation in relation to a host of floral scents by Acalymma vittatum (striped cucumber beetles) (Fuentes et al., 2013; Pinto et al., 2007a, b).
In this research, the effects of ozone on volatile-mediated communication between plants, using cabbage/broccoli-P.brassica-P. xylostella as plant-herbivore model system was studied.
A series of laboratory and field experiments under ambient and elevated ozone concentrations was conducted in order to assess the effects of plant-plant signalling among conspecific or heterospecific plants on the susceptibility of HIPV-receivers to oviposition by pest Lepidoptera.
The main aim of this research was to assess how plant-plant signalling helps the receiver plant to be less susceptible to oviposition by the pest Plutella xylostella. A further aim was to assess whether increased levels of ozone eliminate or reduce the effect.
2. LITERATURE REVIEW
2.1. VOLATILES RELEASE IN RESPONSE BIOTIC AND ABIOTIC STRESSES
In response to herbivore damage, plants produce a variety of volatile organic compounds known as herbivore-induced plant volatiles (HIPVs) (Campos, et al., 2008), which differ both qualitatively and quantitatively, according to the species of herbivore (Blande et al., 2010).
HIPVs consist of a blend of terpenes, green-leaf volatiles, aromatic compounds and others (Rodriguez-Saona et al., 2012). During growth, plants produce about 100 000 chemical products out of which 1700 of them are known to be volatile (Dicke and Loreto, 2010.) These volatiles represent 1 % of the known secondary metabolites of plants to date (Dudareva et al., 2006). VOCs are released by various plant vegetative tissues into the atmosphere and from roots into the soil (Robert et al., 2013). The main functions of these volatiles are to protect plants from damage caused by pathogens and herbivores, and to serve as signals in communication between plants (Dudareva et al., 2006).
Different stresses (abiotic and biotic) induce plants to emit a range of volatiles quantitatively and qualitatively (Holopainen, 2004). Abiotic factors (drought, heat stress and ozone) have a strong impact on plant metabolism. O3 reacts rapidly with many VOCs in the atmosphere (Atkinson and Arey, 2003), leading to degradation of some volatiles,including terpenoids and green-leaf volatiles (GLVs) (Pinto et al., 2007). In addition, O3 can affect the VOC emissions by impacting the plant physiology and biochemistry, and by reacting with plant surfaces (Rao et al., 2000; Fruekilde et al., 1998). VOC emissions released to the atmosphere occur by diffusion from leaves. Therefore, the temperature also affects the intensity of emission by increasing the volatility and diffusion coefficients (Niinemets et al., 2004). Biotic factors include pathogens and herbivore feeding. Plants cannot run away from herbivores and cannot avoid adverse effects of the climate, atmosphere, or soil by going somewhere else. They have to acclimate and adapt in order to survive (Becker et al., 2015).
Plants have two types of volatile response depending on when herbivores start to feed. The first response is the rapid emission of stored compounds. These compounds are emitted when plant tissue is damaged. The second response is the de novo synthesis of compounds (Paré et al., 1997). They are not stored, but released by plants as they are produced. The amounts of emitted compounds increase depending on herbivore feeding (Holopainen, 2004).
2.1.1. Role of volatiles as direct and indirect defences of plants
HIPVs play roles in direct and indirect defence; in direct defence, HIPVs can have toxic effects and can repel or deter herbivores (Arimura et al., 2009). Direct defences are mediated by plant characteristics that influence the biology of herbivores, such as mechanical protection on plant surfaces (such as hairs, trichomes, spines, thorns and thicker leaves) or the production of toxic chemicals (such as alkaloids, terpenoids, cyanogenic glycosides, glucosinolates and phenols), which either kill or inhibit the herbivores development (Agrawal et al., 2009; Hanley et al., 2007).
Indirectly, HIPVs affect herbivores by attracting parasitoids, predators and other natural enemies that feed on their eggs and larvae (Kessler and Baldwin, 2001). These indirect defences mediate herbivore-enemy interactions by increasing the inhibition of herbivores and potentially increasing plant fitness (Romero and Koricheva, 2011; Schmitz, Hamback and Beckerman, 2000). Both defences can be either constitutive, meaning that they are always expressed or inducible, which means that they occur only when necessary, after herbivore damage (Arimura et al., 2005).
There are a large number of chemical compounds that are involved in signalling to herbivores, predators and other natural enemies. Usually many of the volatile compounds effective in communication between plants are the compounds synthesized de novo upon herbivore attack. Several compounds, such as terpenoids and GLVs are known to function in within-plant and plant-plant interactions (López-Larrea, 2012).
Terpenoids are the secondary compounds, which include isoprene, monoterpenes, sesquiterpenes, homoterpenes and diterpenes. Isoprene protects plants from high temperature and ozone stress (Holopainen, 2004). Monoterpene is shown to protect plants against heat stress and can quench ozone in plant tissues (Laothawornkitkul et al., 2009; Holopainen, 2004). In addition, many monoterpenes are involved in plant-plant signalling (Godard et al., 2008; Arimura et al., 2000). Sesquiterpenes plays role in the attraction of pollinators and seed dispensers to fruits (Knudsen et al., 2006). Homoterpenes are known to attract predators and parasitoids and to induce defence reactions in undamaged neighbouring plants (Kappers et al., 2005; Arimura et al., 2000).
Green‑leaf volatiles (GLVs) comprise a variety of C6 compounds including alcohols, aldehydes and esters (Matsui, 2006). GLVs are formed via the lipoxygenase pathway and include (Z)-3-hexenal, (Z)-3-hexenyl acetate and (Z)-3-hexen-1-ol, which important signals involved in inducing defence responses in intact neighbouring plants. GLVs are released immediately after a leaf is ruptured by mechanical damage as well as herbivore feeding (Matsui, 2006; Hatanaka, 1993).
2.1.2. Role of volatiles in plant-plant signalling
The role of volatiles in plant-plant signalling has been one of the most interesting and controversial issues in modern ecology (Figure 1). One of the actual points of controversy has been the distance at which HIPV signals can be received by plants (Frost et al., 2008). The distances at which the volatiles can affect other plants depend on abiotic factors, such as air temperature, humidity and wind speed (Heil and Karban, 2010). Karban et al. (2006) and Karban et al. (2003) demonstrated communication between plants under field conditions and observed that the process is effective only over short distances. Sagebrush, for example, releases large amounts of methyl jasmonate and can affect other plants at distances of no more than 60 cm (Karban et al., 2006; Farmer and Ryan, 1990).
Different mechanisms of communication between plants have been activated via VOCs (Rodriguez-Saona et al., 2009). When herbivores start feeding on a plant, several defence- signalling pathways are induced, leading to a variety of protective responses (Fürstenberg- Hägg et al., 2013). VOCs released from damaged leaves come into contact with other leaves of the same plant and with neighbouring plants (Dudareva et al., 2006).
It is important to note, that plants have the ability to distinguish between herbivory and mechanical damage, as well as oviposition. This ability is necessary to avoid the loss of costly defence resources as the production and release of defence metabolites only benefits herbivore infested plants (Fürstenberg-Hägg et al., 2013). Intraspecific interplant signalling by HIPVs has been shown for some species, including lima bean, tobacco, maize, cotton, poplar and willow, while tomato and tobacco have been shown to increase defence responses when exposed to damaged sagebrush volatiles, which are examples of interspecific signalling (Heil and Karban, 2010).
Figure 1. Schematic representation of within-plant and plant-plant signalling via volatile organic compounds (VOCs) (Photo: Girón-Calva S.).
A signalling emitter plant releases volatiles, which not only reduces the quantity of attacking herbivores on the emitting plant, but can also reduce herbivore attack on the neighbouring plants (Kessler and Baldwin, 2001; Shulaev et al., 1997). This signalling induces emission of volatiles in neighbouring plants (Dudareva et al., 2006). VOCs can be repellent signals for generalist herbivores, but specific types of volatile signals are emitted by a plant individual, which gives information on the plant identity. If VOCs received by specialist herbivores will increase feeding damage, the plant’s fitness will be decreased (Bruce et al., 2005).
2.1.3 Active and passive plant-plant interactions
In response to herbivore damage, plants release specific blends of volatiles (Dicke and Baldwin, 2010). The plant volatile compounds provide chemical information about the attack status of the emitting plant, which can be detected by neighbouring-receiver plants (Kost and Heil, 2006). Plants receive volatile compounds emitted by damaged neighbouring plants and acquire an increased level of resistance to herbivores (Li and Blande, 2015; Sugimoto et al., 2014). This resistance can be divided into active (detection of volatiles) and passive (adsorption of volatiles) categories, both of which occurs in the passage of VOCs between plants (Li and Blande, 2015; Choh et al., 2004).
The active plant-plant interaction involves reception of a chemical signal and a physiological change in receiver plants (Li and Blande 2015). Receiver plants may change gene expression in response to herbivory (Peng et al., 2011; Arimura et al., 2000). VOCs can induce the defence gene expression directly and may enhance resistance of receiver plants (Ton et al., 2006; Engelberth et al., 2004).
One of the most frequently observed responses of receiver plants is a priming of defences, whereby the plant does not activate defences immediately, but is prepared to respond rapidly and intensely if attacked. VOCs, for example, released by maize infested with Spodoptera littoralis caterpillars primed their neighbours, which reacted with a rapid and stronger response to successive herbivore attack (Ton et al., 2006). The VOCs activated the JA- pathway regulating certain defence genes responsible for induction of both direct and indirect defence mechanisms in the primed plants (Ton et al., 2006; Engelberth et al., 2004; Arimura et al., 2000). They emitted the GLVs, (Z)-3-hexenal, (Z)-3-hexen-1-ol or (Z)-3-hexenyl acetate and increased their attractiveness to parasitic Cotesia marginiventris wasps. As a result, they were damaged less by the caterpillars (Ton et al., 2006; Engelberth et al., 2004).
The passive plant-plant interaction includes volatiles from emitter plants adsorbing to the surfaces of the receiver plant without changing the plant other than chemical changes to the surface (Li and Blande, 2015). VOCs can be adsorbed to and re-released from conspecific plants (Choh et al., 2004; Himanen, 2010), for example, undamaged Lima bean (Phaseolus lunatus) plants exposed to spider mite (Tetranychus urticae)-infested Lima bean plants released volatiles associated with herbivore damage. Receiver plants could not synthesize these volatiles and it was concluded that adsorption of the chemicals to the leaf surfaces had occurred. The passive adsorption and re-release of volatiles has also been shown in interspecific interactions under field conditions (Himanen, 2010).
Many volatiles play a role as mediators of plant–plant signalling (Pinto et al., 2010). Blande et al. (2010) demonstrated that elevated ozone levels significantly reduce the distance of active plant-plant communication in lima bean. This leads to degradation of volatile signals between emitter and receiver plants (Li and Blande, 2015). In addition, GLVs and terpenes are known mediators of plant-plant signalling (Godard et al., 2008; Kost and Heil, 2006;
Farag and Pare, 2002).
Exposure to ozone can cause either induction or reduction of GLV and terpenes emissions (Hartikainen et al., 2012; Pellegrini et al., 2012; Himanen et al., 2009; Vuorinen et al., 2004).
Many experiments demonstrated that increasing levels of ozone could have significant effects on interactions mediated by volatiles (Li and Blande, 2015; Blande et al., 2011; Blande et al., 2010).
2.2 EFFECT OF OZONE ON PLANTS
Ozone (O3) is a secondary air pollutant produced by the reaction of nitrogen oxides (NOx) with methane (CH4), carbon monoxide (CO), or volatile organic compounds (VOCs) under sunlight (The Royal Society, 2008; Fowler et al., 1999). O3 is found in both the stratosphere and the troposphere.
Stratospheric ozone is not anthropogenic and it protects the earth from the sun's harmful UV radiation. Therefore, stratospheric ozone is known as “good O3”. Tropospheric ozone is mostly derived from anthropogenic activities (Ainsworth et al., 2012), and is a major component of photochemical smog and can be referred to as “bad O3”. The VOCs and NOx,
which form smog are emitted by power plants, biomass burning and motor vehicles (The Royal Society, 2008). Tropospheric ozone negatively influences the health of people and natural ecosystems. Tropospheric ozone is also a greenhouse gas (Shindell et al., 2009).
Over the last century, ambient levels of ozone have more than doubled (Stevenson et al., 2000). Concentrations of ozone are higher in the northern hemisphere than the southern hemisphere with mean monthly O3 mixing ratios in the northern hemisphere ranging from about 35-50 ppb (Stevenson et al., 2006). Ozone levels are greatest in summer and can rise as high as 80-200 ppb (Fowler et al., 1999). Ozone pollution is expected to increase over the next hundred years. The background ozone concentration in the northern hemisphere is projected to reach 50 ppb by 2050 and 60 ppb by 2100 (Wittig, Ainsworth and Long, 2007).
Ozone is considered one of the most dangerous oxidant molecules for plants (Ashmore, 2005). Over the last years, many researchers have studied the effects of ozone on plants, especially focusing on plant cells and tissues, net photosynthesis, visible injuries, growth and development of different kinds of plants (Yendrek et al., 2013; Pinto et al., 2010; Ashmore,
2005). The harmful effects of O3 on plants vary and depend on the concentration of ozone, and the genetics and varieties of the plant (Heck and Miller, 1994), for example, among herbaceous plants tobacco is known to be especially sensitive to ozone. Tobacco cultivars including ozone-tolerant Bel B and supersensitive Bel W3 have been used as distinctive bio monitors of ozone (Pasqualini et al., 2001).
The highest amount of O3 uptake into the leaves occurs during the plant photosynthesis when stomata are open. Ozone enters into leaves through their stomata and reacts in the apoplast of the mesophyll cells, producing reactive oxygen species (ROS) (Ainsworth et al., 2012). These radicals induce a series of signalling pathways and plant defence responses, which results in damage to mesophyll cells (Chen et al., 2009). Leaves exposed to ozone often have visible symptoms such as stippling, chlorosis, and necrosis (Bell et al., 2002; Pell, 1997). Usually symptoms of necrosis are evident between the veins on the top surface of a leaf. The severity and type depends on some factors, such as duration and concentration of ozone, weather conditions and genetics of the plant (Fares et al., 2009).
The symptoms of ozone damage are divided into two groups: acute and chronic. Both acute and chronic exposures have a strong damage to plants with relatively different symptoms.
Acute ozone stress means short-term ozone exposure with daily concentrations 120-500 ppb (Kangasjärvi et al., 2001).
Acute exposure to ozone causes similar reactions to hypersensitive response (HR) (Rao et al., 2000).This leads to visible necrotic lesions and induction of a programmed cell death in most sensitive plants (Pinto, 2008). Programmed cell death (PCD) is controlled by phytohormones:
salicylic acid, ethylene and jasmonic acid (Baier et al., 2005; Kangasjärvi et al., 2005). At first, salicylic acid is produced and participates in the initiation of damage or the formation of the hypersensitive response. After several hours, ethylene biosynthesis is activated, and it is involved in damage distribution. Then jasmonic acid is synthesized and collects on borders of the damage (Pinto, 2008).
Chronic ozone stress occurs slowly over a longer time with daily ozone concentrations of 40- 120 ppb (Rao et al., 2000). Chronic stress may occur with or without visible damage.
Sometimes the only symptoms of chronic exposure are decreases in photosynthetic level, plant productivity and premature senescence (Pasqualini et al., 2001). Visible damage
resulting from chronic exposure includes variations in bronzing or pigmentation, chlorosis and necrosis after chronic exposure to low ozone concentrations (Felzer et al., 2007). In addition, chronic exposure to sensitive plants may cause changes in the plant physiology, for example, leaves grow slower than normally, and also older leaves are characteristically more affected than young leaves to ozone stress (Freiwald et al., 2008).
Tropospheric ozone and other reactive species in the atmosphere reduce plant-plant interaction by degrading many VOCs. Blande et al. (2010) in their experiment have observed that elevated ozone concentration can significantly decrease the distance of plant-plant signalling in lima bean, in another experiment it has also been shown that ozone affects the efficiency of feeding by arthropods and orientation in relation to a host of floral scents by striped cucumber beetles (Acalymma vittatum) (Fuentes et al., 2013; Pinto et al., 2007a, b).
2.2.1 Effects of ozone on VOCs and plant-plant signalling
In recent years, many researchers have been interested in estimating the atmospheric and environmental impacts of ozone on VOC emissions (Pinto et al., 2010). Ozone reacts with many VOCs in the atmosphere, which leads to rapid degradation of certain volatiles and may consequently affect the VOC-mediated signalling (Blande et al., 2010). The atmospheric lifetimes of VOC emissions are relevant to define how effective they will be in mediating interactions. Volatile organic compounds, such as GLVs, monoterpenes and sesquiterpenes have lifetimes in the atmosphere, which are only a few minutes and hours or less than 24 hours (Yuan et al., 2009; Erb et al., 2009). Other VOCs that are less reactive with atmospheric oxidants have atmospheric lifetimes longer than 24 hours. The high reactivity and short atmospheric lifetimes substantially reduce the signalling distance of most reactive compounds (Yuan et al., 2009).
After acute, and in certain cases after chronic exposure, increased or induced VOC emissions have been a common response (Rao et al., 2000), for example, O3 has been observed to induce the synthesis of MeSA and increase the emissions of sesquiterpenes in both O3- sensitive and O3-tolerant cultivars of tobacco plants (Nicotiana tabacum L.) (Beauchamp et al., 2005; Heiden et al., 1999). In addition, the homoterpene (E)-4,8-dimethyl-1,3,7- nonatriene (DMNT) in Lima bean plants is commonly emitted by herbivore damaged plants and is also induced by acute O3 exposure (Vuorinen et al., 2004).
One of the issues associated with airborne plant-plant signalling is the distance over which signals are efficient. The distances at which the signal chemicals can operate are limited by abiotic factors (Blande et al., 2011). In experiments of Blande et al. (2010) it was demonstrated that ozone of an 80 ppb concentration significantly decreased the distance over which interaction between plants was effective. Several experiments demonstrating plant- plant signalling have shown effectiveness only at short distances for example, signal emitting from sagebrush (Artemisia tridentata Nutt.) plant to tobacco (Nicotiana attenuata Torr.) plant has been effective at short distances (10 cm) (Karban et al., 2003). In other experiments, signal emitting from Artemisia tridentata plant to other Artemisia tridentata plants has been demonstrated at 60 cm distances (Karban et al., 2006).
2.3 BRASSICA OLERACEA L. VAR. CAPITATА (CABBAGE) AND BRASSICA OLERACEA VAR. ITALICA (BROCCOLI) AS STUDY SYSTEMS FOR FIELD AND LABORATORY EXPERIMENTS
The Brassica genus belongs to the mustard family Brassicaceae, consisting of about 375 genera and 3200 species worldwide (Ahuja et al., 2010; LeCoz and Ducombs, 2006).
Brassica comprises about 100 species, which includes cabbage, broccoli, Brussels sprouts, cauliflower and different weeds (Gomez Campo, 1999). This genus of Brassica is known as more important agricultural and horticultural crops and cultivated in most parts of the world (Purty et al., 2008; Hong et al., 2008).
Many of the wild species of Brassica grow as weeds, particularly in North America, South America and Australia (Purty et al., 2008). Different species are popular for their significant nutritional and economic value. They are grown for oil, spices, vegetables or animal feed (Ashraf and McNeilly, 2004). Cabbage (Brassica oleracea L. var. capitata) is a source of protein with high biological value and its leaves are rich in vitamin A, B1, B2, C and minerals (Sarikamiş et al., 2009). The economic value of cabbages is decreased when their leafy heads crack or split, also when they are damaged by insects (Pang et al., 2015). Broccoli (Brassica oleracea var. italica) is a thermosensitive crop. Broccoli usually needs cool temperatures for optimum growth (Narendra et al., 2007). It is rich in health promoting phytochemicals, such as vitamin A, C and glucosinolates. In addition, broccoli is one of the most widely consumed vegetables in China (Xu et al., 2006).
Brassicaceae family contains a group of specific secondary metabolites, the glucosinolates (mustard oil glucosides) (Bones and Rossiter 2006; Fahey et al., 2001). The glucosinolates represent a large group of non-volatile and sulphur-containing secondary metabolites, which takes place in all Brassica crops (Tripathi and Mishra, 2007). About 120 different glucosinolates have been identified to date (Dekker et al., 2009). The glucosinolate composition and content generally varies depending on the Brassica species (Tripathi and Mishra, 2007; Font et al., 2005).
When plant tissues and cells are damaged, chemically stable glucosinolates hydrolysed by the enzyme myrosinase, and as a result, degradation of products occurs, such as nitriles, isothiocyanates, epithionitriles, thiocyanates and oxazolidines (Bones and Rossiter, 2006).
Glucosinolates have repellent or toxic effects, by which they create a significant mechanism of defence against diseases and pests (Zukalová and Vašák, 2002; Mithen, 1992). In response to herbivore feeding, the concentration of glucosinolate can increase. The high level of glucosinolates can influence both specialist and generalist herbivores, and glucosinolates can be equally effective as stimulants and deterrents (Gols et al., 2008; Kusnierczyk et al., 2008, 2007; Agrawal and Kurashige, 2003; Li et al., 2000; Rask et al., 2000).
2.4 LEPIDOPTERA PESTS 2.4.1 Pieris brassicae
The large cabbage white butterfly, Pieris brassicae (Figure 2) is a serious pest of many brassicaceous crops in Europe, North Africa and Asia, causing defoliation of plants and reducing the growth capacity (Johnson and Triplehorn, 2005). P. brassicae caterpillars feed only on glucosinolate-producing plants, such as the Brassicaceae family, including cabbages and mustards (Hopkins et al., 2009).
It leads to damage at all stages of growth and can bore the head of cabbage, with high numbers of P. brassicae destroying plant leaves and completely defoliating and killing plants (Hasan and Ansari, 2010; Hasan, 2008; Lal and Ram, 2004; Siraj, 1999). The consumption rate of each larva is about 74 to 80 cm² leaf area (Younas et al., 2004). Females lay their eggs on the downside of leaves of host plants (Le Masurier, 1994).
The damaged crops are not harvestable and additionally become infested with bacteria and fungi (Zibaee, 2012). The most common control strategies on P. brassicae are chemical insecticide-based approaches. However, exceeding use of insecticides has harmful effects on human health, environment and natural ecosystems. There is interest in using effective alternative methods to control the large white butterfly and reduce its damage on crops (Hamshou, 2010).
Figure 2. Pieris brassicae, A: female, B: egg clutch, C: caterpillar (Photo A: Holopainen J.;
Photo B: Blande J.; Photo C: Holopainen J.).
2.4.2 Plutella xylostella
The diamondback moth (Plutella xylostella) is a migratory insect (Figure 3), and is known as one of the most serious insect pests of cultivated Brassicaceae plants (Sarfraz, 2006; Talekar and Shelton, 1993). P. xylostella feeds on plants of the Brassicaceae family that contain mustard oils and their glucosides (Talekar and Shelton, 1993). It is adapted to different climatic conditions and causes serious economic losses worldwide (Jankowska and Wiech, 2006; Martínez-Castillo et al., 2002).
The diamondback moth was first noticed as a pest in South Africa in the early 1900s, but is now present worldwide wherever its host plants exist (Shelton, 2001; Charleston and Kfir, 2000). It can damage plants at all stages of growth and female moths lay their eggs singly or in groups on the underside of leaves. Larval chewing makes holes in leaves and can make leaves appear “windowpaned”, with a clear cuticle left after feeding (Ahuja et al., 2010).
As is known in many countries, the diamondback moth has developed resistance to almost all synthetic insecticides used against it, such as the insecticide based on Bacillus thuringiensis Berliner (Bt) formulations (Liu et al., 1995; Tabashnik et al., 1990). The wide usage of Bt products has led to an increasing number of reports of field resistance by P. xylostella (Tabashnik, 1994). As existing insecticides become useless, new insecticides are continuously being developed, but P. xylostella has developed resistance very quickly to many of these (Nisin et al., 2000; Shelton et al., 2000).
Figure 3. Plutella xylostella, A: adults, B: eggs, C: caterpillar (PhotoA, B, C: Holopainen J.).
3. OBJECTIVES
The main aim of this research was to determine:
1. The effect of volatile mediated plant-plant signalling on the susceptibility of cabbage receiver plants to oviposition by the specialist pest Plutella xylostella.
2. Whether elevated levels of ozone eliminate or reduce the plant-plant signalling and alter the herbivore oviposition choices.
3. Whether broccoli emitter plants had a different effect on the susceptibility of cabbage receiver plants to oviposition.
4. MATERIALS AND METHODS
4.1 PLANTS AND INSECTS
Brassica oleracea var. capitata cv. Lennox (cabbage) and Brassica oleracea var. italica cv.
Lucky (broccoli) seeds were sown in 1-L pots in a mixture of peat, soil and sand (3: 1: 1), grown for 4 weeks under greenhouse conditions and after transferred to chambers (Weiss Bio 1300; Weiss Umwelttechnik Gmbh, Reiskirchen-Lindenstruth, Germany) [Day 16L (photosynthetically active radiation 300 µmol ˉ² s ˉ¹), 23 °C, 60% humidity: Night 8D, 18 °C, 80% humidity] for laboratory experiments or transferred to the field for field experiments.
Plutella xylostella and Pieris brassicae were reared in an insectary with a temperature ranging from 20 to 25°C and a photoperiod of 16L: 8D. Larvae were reared on broccoli and Brussels sprouts plants, respectively. Adults of P. xylostella were provided with water and an approximately 30% honey solution, both soaked into cotton wool. Third-instar larvae were used to damage plants and 3-4 day old females of P.xylostella were used in oviposition experiments.
4.2 FREE AIR CONCENTRATION ENRICHMENT (FACE) FACILITIES
The FACE facilities of the UEF are located at Ruohoniemi (62º 13’N, 27º 35’E, 80 m.s.l.). It consists of four ambient-ozone control plots and four enriched-ozone plots (Figure 4). Ozone is produced from pure oxygen by an ozone generator (G21, Pacific Zone Technology Inc., Brentwood, CA, USA) and then released into exposure rings through vertical vent pipes.
Concentrations of ozone were monitored in the centre of the ozone exposure rings at a height of 1.5 m with three UV photometric ozone analysers (Model 1008-RS, Dasibi Environmental Corp., Glendale, CA; Model O3 42 Module, Environment S.A., Poissy). In addition, each plot was constantly monitored for wind speed and direction, so that the vents opened according to wind direction and resulted in ozone flowing into the plots (Anemometer A100; Windvane W200, Vector Instruments).
Figure 4. A: View of the FACE (Free Air Concentration Enrichment) facilities of the UEF at Ruohoniemi from the air. B: View of an enriched-ozone plot (Photo A, B: Holopainen J.)
4.3 CONTROLLED ENVIRONMENT CHAMBERS AND OZONE EXPOSURE SYSTEM
Four Weiss 1300 Bio chambers (75 cm W; 128 cm L; 130 cm H) (Weiss Umwelttechnik Gmbh, Reiskirchen-Lindenstruth, Germany) were used for the experiments. Illumination in these chambers was LED-based. All chambers had a light: dark cycle of 16L: 8D, temperatures of 23°C during the light period and 18°C during the dark period. Relative humidity is 55% during the day and 80% during the night.
The chambers were equipped with an ozone exposure system and had one-directional vertical laminar air flow through a fabric wall. Ozone was produced from pure oxygen with a Fischer Ozone 500 ozone generator (Fischer, Bonn, Germany) and passed through Teflon tubes to the control unit. The control unit regulated ozone flow into each chamber. The entire system was controlled by computer.
Figure 5. Inside view of the controlled environment chambers for HIPV exposure (Photo: Girón-Calva S.)
4.4 PLANT-PLANT COMMUNICATION BETWEEN CABBAGE PLANTS UNDER FIELD CONDITIONS AND OVIPOSITION PREFERENCE TEST OF P.
XYLOSTELLA
4.4.1 Phase 1 Exposure to HIPVs
The first phase of the field experiment was an exposure of receiver plants to volatiles emitted by differently treated emitter plants. The emitter and receiver plants were arranged in cages (60 x 33 x 33cm). The emitter plants were either infested with third instar P. brassicae larvae, or were non-infested controls. In the middle of the cage (between emitter and receiver plants) there was a mesh partition to prevent the movement of the larvae between plants (only for infested plants). The distance between emitter and receiver plants was 30cm.
The experiment was set up in exactly the same way in two elevated and two ambient-ozone plots. The mixing ratio of ozone in the elevated ozone plots was regulated to be 1.5 times the ambient ozone concentration. This phase was maintained for 3 days and plants were watered every day. At the end of the exposure period the receiver plants were removed and paired in oviposition assays (phase 2).
Figure 6. Field set-up for HIPV exposure (Photo: Malikova G. and Girón-Calva S.)
4.4.2 Phase 2 Oviposition preferences of P. xylostella
In the field, receiver plants were labelled and paired in cages (60 x 33 x 33cm) for oviposition choice tests. The plants were positioned 30cm apart. The choice tests were conducted in ambient and elevated-ozone plots and fifty P. xylostella adults were released into each cage.
Adult moths were provided with water and an approximately 30% honey solution, which was soaked into cotton wool. Oviposition tests were maintained for 48 hours, after which the number of eggs deposited on each leaf was counted. The experiment was conducted eleven times.
4.5 PLANT-PLANT COMMUNICATION BETWEEN CABBAGE PLANTS UNDER LABORATORY CONDITIONS AND OVIPOSITION PREFERENCE TEST OF P.
XYLOSTELLA
4.5.1 Phase 1 Exposure to HIPVs
This experimental set up was maintained in two chambers with an ambient-ozone level of approximately 10 ppb and two chambers with an enriched-ozone level of 80 ppb. Plants were placed in controlled chambers in two parallel rows of four plants which were positioned with a gap of 30cm between emitters and receivers. The first row of plants (right side) was emitter cabbage plants, which were infested with 24 third instar P. brassicae larvae. A second row of plants (left side) was receiver cabbage plants. Air within the chambers flowed from right to left. To prevent the movement of larvae from treated emitter to receiver plants the plants were
enclosed in mesh cages. Also exposure to HIPVs experiment was done for control plants. The experiment for controlled plants was set up in the same way, but without larvae. Chambers were controlled by computer with a light: dark cycle of 16L: 8D and temperatures of 23° C.
The first phase of the laboratory experiment was maintained for 3 days and repeated five times.
4.5.2 Phase 2 Oviposition preferences of P. xylostella
The oviposition preferences of P. xylostella were examined in polycarbonate cages (0.7 x 0.7 x 1m), with a fabric mesh window at each end. Receiver plants were taken out of the chambers and labelled according to the treatment ambient control receiver (AmbCR), ambient damage receiver (AmbDR), ozone control receiver (OzoCR) and ozone damage receiver (OzoDR).
Labelled plants were placed in a cage and positioned such as control receivers placed opposite to infested receiver plant and released one hundred P. xylostella adults into each cage (used two cages). Adults of P. xylostella were provided with water and an approximately 30%
honey solution, both soaked into cotton wool. The oviposition assay was run for 48 hours and the number of eggs laid on each plant leaf was counted under a microscope. This experiment was repeated ten times.
Figure 7. A multiple choice cage for oviposition preferences of P. xylostella (Photo: Girón-Calva S.)
4.6. PLANT-PLANT COMMUNICATION BETWEEN BROCCOLI AND CABBAGE PLANTS UNDER LABORATORY CONDITIONS AND OVIPOSITION
PREFERENCE TEST OF P. XYLOSTELLA
4.6.1 Phase 1 Exposure to HIPVs
For this experimental set-up, four chambers were used, two of which had an ambient-ozone concentration of approximately 10 ppb and two an enriched-ozone concentration of 80 ppb.
Plants were placed in plastic trays in two parallel rows of four, which were positioned with a distance of 30cm between the stems of receiver and emitter plants. Infested plants were enclosed in mesh cages, to prevent the movement of larvae from emitter to receiver plants.
The first row of plants was emitter broccoli plants, which were infested with 24 (6 larvae per plant) third instar P. brassicae larvae. A second row of plants was receiver cabbage plants.
Air within the chambers flowed from right to left. For the control plants, the same experimental set-up was used, the only difference was that plants were placed in open plastic trays and were not infested with larvae. The experiment was maintained for 3 days and conducted nine times.
4.6.2 Phase 2 Oviposition preferences of P. xylostella
Oviposition choice experiments were conducted in polycarbonate cages (0.7 x 0.7 x 1m) with a fabric mesh window at each end. The cages were placed in a controlled environment room with a temperature of 20°C. Receiver plants were labelled according to the treatment ambient control receiver (AmbCR), ambient damage receiver (AmbDR), ozone control receiver (OzoCR) and ozone damage receiver (OzoDR).
Treatments were placed in a cage with position control receiver plants placed opposite to infested receiver plants. One hundred P. xylostella adults were released into each cage and were provided with water and an approximately 30% honey solution soaked into cotton wool.
This experiment was continued for 48 hours, after which the number of eggs laid on each plant was counted. The process was repeated seventeen times.
4.7 DATA ANALYSIS
All statistical analyses were conducted with the software package IBM SPSS 21.0. The percentage of eggs deposited on plants, in both laboratory and field experiments were checked for normality using the Shapiro-Wilk test. The level of significance was selected as 𝛼=0.05.
Paired-sample t-tests were used to test the percentage of eggs deposited on plants in the field experiment. The percentage of eggs laid by P. xylostella on plants exposed to the different treatments under laboratory conditions were compared by univariate ANOVA.
5. RESULTS
5.1 PLANT-PLANT COMMUNICATION BETWEEN CABBAGE PLANTS UNDER FIELD CONDITIONS AND OVIPOSITION PREFERENCE TEST OF
P. XYLOSTELLA
In field experiments, exposure to herbivore-induced plant volatiles from cabbage plants decreased the susceptibility of cabbage receiver plants to oviposition by P. xylostella under ambient ozone conditions, but not when ozone levels were elevated. The percentage of eggs laid on plants exposed to undamaged plants was significantly greater than on plants exposed to damaged plants at ambient ozone levels (t = 2.277; df = 10; P = 0.046). The effect is impaired under elevated ozone conditions, where exposure to damaged neighbours had no significant effect (t = - 1.061; df = 10; P = 0.314) (Figure 8).
Figure 8. The mean percentage (%) of eggs deposited on receiver plants exposed under ambient (10 ppb) and elevated (80 ppb) ozone conditions (mean ±SE, n=11).
Control VOC (cr-VOC) defines plants exposed to undamaged plants (under ambient and elevated ozone). Ir-VOC denotes plants exposed to herbivore-induced plants (under ambient and elevated ozone). A paired-sample t-test was used to test for statistical significance of differences between cr- VOC and ir-VOC plants (α=0.05). P-values are for paired t-tests between cr-VOC and ir-VOC treatments at both ozone (ambient, elevated) levels.
0%
20%
40%
60%
80%
100%
Oviposition (%)
Ambient ozone
cr-VOC ir-VOC
P = 0.046 t-test
0%
20%
40%
60%
80%
100%
Oviposition (%)
Elevated ozone
cr-VOC ir-VOC
P = 0.314 t-test
5.2 PLANT-PLANT COMMUNICATION BETWEEN CABBAGE PLANTS UNDER LABORATORY CONDITIONS AND OVIPOSITION PREFERENCE TEST OF P. XYLOSTELLA
The results from laboratory experiments showed that exposure to herbivore-induced plant volatiles from cabbage plants tends to decrease the susceptibility of cabbage receiver plants to oviposition by P. xylostella in ambient ozone conditions. In Figure 9, P. xylostella females laid marginally more eggs on control cabbage receivers than on herbivore-induced cabbage receivers. The deposited eggs percentage on control receivers about 15% (±0.99 SE) and 13%
(±1.04 SE) of eggs deposited on herbivore-induced receivers under ambient ozone conditions.
Although ozone itself seems to overshadow the plant-plant interactions effect under elevated ozone conditions since both Ozo-cr-VOC and Ozo-ir-VOC are less susceptible to oviposition by P. xylostella. Females deposited 11% (±1.11 SE) of the eggs on control cabbage receivers and the same number of eggs deposited 11% (±1.30 SE) on herbivore-induced cabbage receivers under elevated ozone conditions (ANOVA: F3=2.769, P=0.056, n=10).
Figure 9. The mean percentage (%) of eggs deposited on receiver plants exposed under ambient (10 ppb) and elevated (80 ppb) ozone conditions (mean ±SE, n=10).
Univariate ANOVA and post hoc LSD test were used for statistical analysis. Different letters on bars mean significant differences.
0%
3%
6%
9%
12%
15%
18%
Oviposition (%)
Amb-cr-VOC Amb-ir-VOC Ozo-cr-VOC Ozo-ir-VOC
P = 0.056 ANOVA
a
ab
b b
5.3 PLANT-PLANT COMMUNICATION BETWEEN BROCCOLI AND CABBAGE PLANTS UNDER LABORATORY CONDITIONS AND OVIPOSITION
PREFERENCE TEST OF P. XYLOSTELLA
Results from the laboratory experiment testing for plant-plant interactions between broccoli emitter and cabbage receiver plants showed that exposure to herbivore-induced plant volatiles from broccoli plants tends to increase the susceptibility of cabbage receiver plants to oviposition by P. xylostella under ambient ozone conditions.
The results shown in Figure 10 demonstrate that P. xylostella females deposited 10% (±0.79 SE) of eggs on control cabbage receivers and deposited 13% (±0.73 SE) of eggs on herbivore- induced cabbage receivers in ambient ozone conditions. Ozone itself seems to overshadow the effect of the plant-plant interaction under elevated ozone conditions since both Ozo-cr-VOC and Ozo-ir-VOC are also more susceptible to oviposition by P. xylostella. Females laid 13 % (±0.85 SE) of eggs on Ozo-cr-VOC receiver cabbage plants and 14% (±0.92 SE) of eggs on Ozo-ir-VOC receiver cabbage plants deposited (ANOVA: F3=2.932, P=0.04, n=17).
Figure 10. The mean percentage (%) of eggs deposited on receiver plants exposed under ambient (10 ppb) and elevated (80 ppb) ozone conditions (mean ±SE, n = 17).
The percentage of eggs in each treatment was compared using univariate ANOVA. Multiple comparisons between each treatment were made with the post hoc LSD test. Same letters on bars means non-significant differences between the treatments.
0%
4%
8%
12%
16%
Oviposition (%)
AmbCR-VOC AmbIR-VOC OzoCR-VOC OzoIR-VOC
P = 0.04 ANOVA
a
b b b
6. DISCUSSION
The aim of this study was to determine the effect of volatile mediated plant-plant signalling on the susceptibility of cabbage receiver plants to oviposition by the specialist pest Plutella xylostella, whether elevated levels of ozone eliminate or reduce the plant-plant signalling and alter the herbivore oviposition choices and finally, whether broccoli emitter plants have a different effect on the susceptibility of cabbage receiver plants to oviposition.
Under ambient ozone levels, plants exposed to herbivore-damaged plants were less susceptible to oviposition by P. xylostella, than undamaged plants under field conditions. In addition, the effectiveness of plant-plant signalling under elevated ozone (80 ppb) conditions was investigated and demonstrated that interactions between cabbage plants were disrupted by ozone. In this case, plants exposed to damaged plants were even observed to be less defended, but it is not statistically significant.
Furthermore, in laboratory conditions, two experiments with conspecific and heterospecific plants were conducted to examine the attractiveness of receiver plants to oviposition by P.
xylostella. In the first experiment, which was done with conspecific plants, plants exposed to herbivore-damaged neighbours were less susceptible to oviposition by P. xylostella than plants exposed to undamaged plants under ambient ozone levels. Also, plants exposed to both herbivore-damaged and undamaged plants under elevated ozone levels had equal resistance to oviposition by P. xylostellа. In addition, the effect of ozone on the attractiveness of receiver plants to oviposition was tested with heterospecific plants. Receiver plants exposed to herbivore-damaged broccoli emitter plants were more susceptible to oviposition than control receiver plants under ambient ozone levels.
This was the opposite to observations with conspecific plants, and again the interaction was broken down under elevated ozone conditions. The reason for this might be that ozone degrades volatiles emitted from the emitter plants before they reach the receiver plants (Blande et al., 2010); therefore the receiver plants cannot detect the signal from emitter plants in the presence of ozone.
Results show that under ambient ozone levels, receiver cabbage plants exposed to herbivore- damaged cabbage plants were better defended from oviposition by P. xylostellа under both
