CHARACTERISATION OF THE TRITROPHIC INTERACTIONS BETWEEN TOBACCO YELLOW DWARF VIRUS , ITS VECTOR AND HOST-PLANTS
by
Piotr Tr ębicki
Centre for Tropical Crops and Biocommodities Faculty of Science and Technology
A thesis submitted for the degree of Doctor of Philosophy at the Queensland University of Technology
2010 ABSTRACT
Tobacco yellow dwarf virus (TbYDV, family Geminiviridae, genus Mastrevirus ) is an economically important pathogen causing summer death and yellow dwarf disease in bean (Phaseolus vulgaris L.) and tobacco ( Nicotiana tabacum L.), respectively. Prior to the commencement of this project, little was known about the epidemiology of TbYDV, its vector and host-plant range. As a result, disease control strategies have been restricted to regular poorly timed insecticide applications which are largely ineffective, environmentally hazardous and expensive. In an effort to address this problem, this PhD project was carried out in order to better understand the epidemiology of TbYDV, to identify its host-plant and vectors as well as to characterise the population dynamics and feeding physiology of the main insect vector and other possible vectors.
The host-plants and possible leafhopper vectors of TbYDV were assessed over three consecutive growing seasons at seven field sites in the Ovens Valley, Northeastern Victoria, in commercial tobacco and bean growing properties. Leafhoppers and plants were collected and tested for the presence of TbYDV by PCR. Using sweep nets, twenty-three leafhopper species were identified at the seven sites with Orosius orientalis the predominant leafhopper. Of the 23 leafhopper species screened for TbYDV, only Orosius orientalis and Anzygina zealandica tested positive. Forty-two different plant species were also identified at the seven sites and tested. Of these, TbYDV was only detected in four dicotyledonous species, Amaranthus retroflexus , Phaseolus vulgaris , Nicotiana tabacum and Raphanus raphanistrum .
Using a quadrat survey, the temporal distribution and diversity of vegetation at four of the field sites was monitored in order to assess the presence of, and changes in, potential host-plants for the leafhopper vector(s) and the virus. These surveys showed that plant composition and the climatic conditions at each site were the major influences on vector numbers, virus presence and the subsequent occurrence of tobacco yellow dwarf and bean summer death diseases. Forty-two
II plant species were identified from all sites and it was found that sites with the lowest incidence of disease had the highest proportion of monocotyledonous plants that are non hosts for both vector and the virus. In contrast, the sites with the highest disease incidence had more host-plant species for both vector and virus, and experienced higher temperatures and less rainfall. It is likely that these climatic conditions forced the leafhopper to move into the irrigated commercial tobacco and bean crop resulting in disease.
In an attempt to understand leafhopper species diversity and abundance, in and around the field borders of commercially grown tobacco crops, leafhoppers were collected from four field sites using three different sampling techniques, namely pan trap, sticky trap and sweep net. Over 51000 leafhopper samples were collected, which comprised 57 species from 11 subfamilies and 19 tribes. Twenty- three leafhopper species were recorded for the first time in Victoria in addition to several economically important pest species of crops other than tobacco and bean. The highest number and greatest diversity of leafhoppers were collected in yellow pan traps follow by sticky trap and sweep nets. Orosius orientalis was found to be the most abundant leafhopper collected from all sites with greatest numbers of this leafhopper also caught using the yellow pan trap.
Using the three sampling methods mentioned above, the seasonal distribution and population dynamics of O. orientalis was studied at four field sites over three successive growing seasons. The population dynamics of the leafhopper was characterised by trimodal peaks of activity, occurring in the spring and summer months. Although O. orientalis was present in large numbers early in the growing season (September-October), TbYDV was only detected in these leafhoppers between late November and the end of January. The peak in the detection of TbYDV in O. orientalis correlated with the observation of disease symptoms in tobacco and bean and was also associated with warmer temperatures and lower rainfall.
To understand the feeding requirements of Orosius orientalis and to enable screening of potential control agents, a chemically-defined artificial diet (designated PT-07) and feeding system was developed. This novel diet
III formulation allowed survival for O. orientalis for up to 46 days including complete development from first instar through to adulthood. The effect of three selected plant derived proteins, cowpea trypsin inhibitor (CpTi), Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA), on leafhopper survival and development was assessed. Both GNA and WGA were shown to reduce leafhopper survival and development significantly when incorporated at a 0.1% (w/v) concentration. In contrast, CpTi at the same concentration did not exhibit significant antimetabolic properties. Based on these results, GNA and WGA are potentially useful antimetabolic agents for expression in genetically modified crops to improve the management of O. orientalis , TbYDV and the other pathogens it vectors.
Finally, an electrical penetration graph (EPG) was used to study the feeding behaviour of O. orientalis to provide insights into TbYDV acquisition and transmission. Waveforms representing different feeding activity were acquired by EPG from adult O. orientalis feeding on two plant species, Phaseolus vulgaris and Nicotiana tabacum and a simple sucrose-based artificial diet. Five waveforms (designated O1-O5) were observed when O. orientalis fed on P. vulgaris , while only four (O1-O4) and three (O1-O3) waveforms were observed during feeding on N. tabacum and the artificial diet, respectively. The mean duration of each waveform and the waveform type differed markedly depending on the food source.
This is the first detailed study on the tritrophic interactions between TbYDV, its leafhopper vector, O. orientalis, and host-plants. The results of this research have provided important fundamental information which can be used to develop more effective control strategies not only for O. orientalis , but also for TbYDV and other pathogens vectored by the leafhopper.
IV Keywords: virus, vector, tobacco, TbYDV, epidemiology, Orosius orientalis, leafhopper , population dynamics, Geminiviridae , artificial diet, lectins, feeding behaviour, EPG.
V PUBLICATIONS
Peer reviewed publications related to this PhD thesis
1. Tr ębicki P, Harding RM & Powell KS. 2009. Antimetabolic effects of Galanthus nivalis agglutinin and wheat germ agglutinin on nymphal stages of the common brown leafhopper using a novel artificial diet system. Entomologia Experimentalis et Applicata 131 , 99-105.
2. Tr ębicki P, Harding RM, Rodoni B, Baxter G & Powell KS. 2010. Seasonal activity and abundance of Orosius orientalis (Hemiptera: Cicadellidae) at agricultural sites in Southeastern Australia. Journal of Applied Entomology 134 , 91-97.
3. Tr ębicki P, Harding RM, Rodoni B, Baxter G & Powell KS. 2010. Vectors and alternative hosts of TbYDV in south-eastern Australia. Annals of Applied Biology 157 , 13-24.
4. Tr ębicki P, Harding RM, Rodoni B, Baxter G & Powell KS. 2010. Diversity of Cicadellidae in agricultural production areas in the Ovens Valley, Northeast Victoria, Australia. Australian Journal of Entomology 49 , 213-220.
Report related to this PhD thesis
1. Tr ębicki P, Powell KS & Baxter G. 2007. Epidemiology and management of leafhopper that transmit a unique virus In: Sustainable tobacco production. Ed. by Baxter G, Hayes G, Harrington A, Harrington J, Sacco G. Department of Primary Industries, Final Report, TBO4001, 132.
VI TABLE OF CONTENTS
ABSTRACT...... II
PUBLICATIONS ...... VI
TABLE OF CONTENTS...... VII
LIST OF FIGURES ...... XII
LIST OF TABLES ...... XV
LIST OF ABBREVIATIONS ...... XVI
STATEMENT OF ORIGINAL AUTHORSHIP ...... XIX
ACKNOWLEDGEMENTS...... XX
CHAPTER 1 AIMS AND OBJECTIVES...... 1
DESCRIPTION OF SCIENTIFIC PROBLEM INVESTIGATED ...... 1
OVERALL OBJECTIVES OF THE STUDY ...... 1
SPECIFIC AIMS OF THE STUDY ...... 2
ACCOUNT OF SCIENTIFIC PROGRESS LINKING THE SCIENTIFIC PAPERS ...... 2
CHAPTER 2 LITERATURE REVIEW ...... 5
THE AUSTRALIAN TOBACCO INDUSTRY ...... 5 Tobacco in Australia – a historical perspective ...... 5 Cultivated tobacco ...... 5 Production systems and management ...... 6 Major diseases of tobacco ...... 7
TOBACCO YELLOW DWARF DISEASE ...... 8 Introduction ...... 8 Etiology ...... 13 Tobacco yellow dwarf virus ...... 13
THE VECTOR OF TBYDV – OROSIUS ORIENTALIS ...... 16
VII Vector taxonomy ...... 16 Vector characteristics ...... 16 Life stages of Orosius orientalis ...... 17 Host-plants and population dynamics ...... 17 Alternative vectors of TbYDV ...... 25
CONTROL STRATEGIES FOR TBYDV...... 25 Vector and disease management ...... 25 Crop rotation ...... 26 Leafhopper management ...... 26
LEAFHOPPER ARTIFICIAL DIETS ...... 27 Leafhopper nutrition ...... 27
LEAFHOPPER FEEDING BEHAVIOUR ...... 30 Electrophysiological techniques ...... 30 Host-plant selection ...... 35 Feeding strategies ...... 35
SUMMARY ...... 35
REFERENCES ...... 36
CHAPTER 3 VECTORS AND ALTERNATIVE HOSTS OF TBYDV IN SOUTH-EASTERN AUSTRALIA ...... 47
STATEMENT OF AUTHORSHIP...... 48
ABSTRACT ...... 49
INTRODUCTION ...... 50
MATERIALS AND METHODS ...... 51 Sampling sites ...... 51 Weather data ...... 52 Leafhopper collection ...... 52 Plant surveys ...... 57 DNA extraction and PCR ...... 58 Statistical analysis ...... 58
RESULTS ...... 59 Leafhopper diversity and abundance ...... 59
VIII Testing leafhoppers for TbYDV ...... 59 Population dynamics of O. orientalis ...... 63 Potential alternative hosts of TbYDV ...... 63 Seasonal detection of TbYDV ...... 68
DISCUSSION ...... 73
ACKNOWLEDGEMENTS ...... 76
REFERENCES ...... 76
CHAPTER 4 DIVERSITY OF CICADELLIDAE IN AGRICULTURAL PRODUCTION AREAS IN THE OVENS VALLEY, NORTHEAST VICTORIA, AUSTRALIA...... 83
STATEMENT OF AUTHORSHIP ...... 84
ABSTRACT ...... 85
INTRODUCTION ...... 86
MATERIALS AND METHODS ...... 87 Study sites ...... 87 Sampling methods ...... 87 Identification ...... 88 Meteorological data ...... 89
RESULTS ...... 89 Leafhopper diversity and abundance ...... 89 Seasonal leafhopper activity ...... 97
DISCUSSION ...... 99
ACKNOWLEDGEMENTS ...... 101
REFERENCES ...... 102
CHAPTER 5 SEASONAL ACTIVITY AND ABUNDANCE OF OROSIUS ORIENTALIS (HEMIPTERA: CICADELLIDAE) AT AGRICULTURAL SITES IN SOUTHEASTERN AUSTRALIA ...... 109
STATEMENT OF AUTHORSHIP ...... 110
ABSTRACT ...... 111
IX INTRODUCTION ...... 112
MATERIALS AND METHODS ...... 113 Study sites ...... 113 Sampling methods ...... 113 Sticky traps ...... 114 Pan traps ...... 114 Sweep nets ...... 115 Leafhopper identification ...... 115 Meteorological data ...... 116 Statistical analysis ...... 116
RESULTS ...... 116 Seasonal activity ...... 116 Trap evaluation ...... 119 Leafhopper population dynamics ...... 119
DISCUSSION ...... 122
ACKNOWLEDGEMENTS ...... 124
REFERENCES ...... 124
CHAPTER 6 ANTIMETABOLIC EFFECTS OF GALANTHUS NIVALIS AGGLUTININ AND WHEAT GERM AGGLUTININ ON NYMPHAL STAGES OF THE COMMON BROWN LEAFHOPPER USING A NOVEL ARTIFICIAL DIET SYSTEM...... 129
STATEMENT OF AUTHORSHIP...... 130
ABSTRACT ...... 131
INTRODUCTION ...... 132
MATERIALS AND METHODS ...... 133 Insect culture ...... 133 Chemicals and materials ...... 134 Artificial diet preparation ...... 134 Feeding trials ...... 138 Statistical analysis ...... 141
RESULTS AND DISCUSSION...... 141
X ACKNOWLEDGEMENTS ...... 147
REFERENCES ...... 147
CHAPTER 7 ELECTROPHYSIOLOGICAL MONITORING OF THE FEEDING BEHAVIOUR OF OROSIUS ORIENTALIS ON AN ARTIFICIAL DIET AND SELECTED HOST-PLANTS...... 155
STATEMENT OF AUTHORSHIP ...... 156
ABSTRACT ...... 157
INTRODUCTION ...... 158
MATERIALS AND METHODS ...... 160 Insect and plant material ...... 160 Data collection ...... 160 Artificial diet and diet chamber ...... 161 Data analysis ...... 163
RESULTS ...... 163 EPG waveforms on bean, a preferred host-plant ...... 163 EPG waveforms on tobacco, a non-preferred host-plant ...... 167 EPG waveforms on an artificial diet ...... 171 Comparison of feeding behaviour on an artificial diet and host-plants .. 171
DISCUSSION ...... 172
ACKNOWLEDGEMENTS ...... 176
REFERENCES ...... 177
CHAPTER 8 GENERAL DISCUSSION AND CONCLUSIONS...... 183
REFERENCES ...... 187
XI LIST OF FIGURES
FIGURE 2.1 HEALTHY (LEFT ) AND TYD-AFFECTED TOBACCO PLANTS (RIGHT ) IN
THE FIELD IN THE OVENS VALLEY ...... 11
FIGURE 2.2 GENOME ORGANIZATION OF THE THREE MAIN GEMINIVIRUS GENERA .
(S OURCE : ROJAS ET AL . 2005)...... 14
FIGURE 2.3 OROSIUS ORIENTALIS , THE COMMON BROWN LEAFHOPPER ...... 19
FIGURE 2.4 AN EXAMPLE OF AN ARTIFICIAL DIET FEEDING CHAMBER FOR SAP -
SUCKING INSECTS (S OURCE : POWELL ET AL . 1993)...... 29
FIGURE 2.5 . THE COMPONENTS OF AN EPG MONITORING SYSTEM . A - COMPONENTS
OF THE PRIMARY CIRCUIT THAT ARE HOUSED WITHIN THE EPG MONITOR . B –
DIAGRAM OF THE PRIMARY CIRCUIT WHERE THE BIOLOGICAL COMPONENTS
ARE MODELLED AS A VARIABLE RESISTOR , RA. OTHER SYMBOLS : RI, INPUT
RESISTOR (FIXED RESISTOR ); VS, VOLTAGE SOURCE (S OURCE : WALKER 2000) ...... 31
FIGURE 2.6 OROSIUS ORIENTALIS ATTACHED VIA SILVER CONDUCTIVE GLUE TO A
GOLD WIRE TO AN EPG INSECT ELECTRODE (NOT SHOWN ) AND FEEDING ON A
PHASEOLUS VULGARIS LEAF ...... 33
FIGURE 3.1 LOCATION OF FIELD SITES (LABELLED A – G) IN NORTH -EAST
VICTORIA , AUSTRALIA , THAT WERE USED IN THIS STUDY ...... 53
FIGURE 3.2 OVERVIEW OF THE SAMPLING METHODOLOGY USED FOR INSECTS
(SWEEP NET ) AND PLANTS (QUADRANT ) AT EACH FIELD SITE ...... 55
FIGURE 3.3 POPULATION DYNAMICS OF OROSIUS ORIENTALIS DURING THREE
GROWING SEASONS (2005/06, 2006/07 AND 2007/08) FROM FOUR SITES (A, B,
C AND D) MONITORED USING SWEEP NETTING . IN THE 2007-08 COLLECTING
SEASON ONLY SITES A AND B WERE STUDIED ...... 64
FIGURE 3.4 PROPORTION OF TOTAL HOST AND NON -HOST -PLANTS FOR OROSIUS
ORIENTALIS FROM FOUR FIELD SITES (A-D) RECORDED DURING 2006/07
GROWING SEASON AS DETERMINED BY QUADRAT SURVEY ...... 70
FIGURE 3.5 CHANGES IN DISTRIBUTION OF VEGETATION TYPE [SENESCED (DRY ),
DICOTYLEDONOUS AND MONOCOTYLEDONOUS ], RAINFALL AND TEMPERATURE
XII DURING ONE GROWING SEASON (2006/2007) ACROSS THE FOUR FIELD SITES (A,
B, C AND D, LINES = AVERAGE TEMPERATURE , BARS = RAINFALL )...... 71
FIGURE 3.6 NUMBERS AND TBYDV STATUS OF OROSIUS ORIENTALIS , AVERAGE
TEMPERATURE AND RAINFALL DURING THREE CONSECUTIVE GROWING
SEASONS . COMBINED DATA FROM ALL FOUR FIELD SITES : A, B, C AND D (LINES
= AVERAGE TEMPERATURE , BARS = RAINFALL )...... 72
FIGURE 4.1 DIVERSITY AND ABUNDANCE OF LEAFHOPPER SPECIES COLLECTED
FROM FOUR SITES , USING SWEEP NET , PAN AND STICKY TRAPS, IN THE OVENS
VALLEY , NORTHEAST VICTORIA , OVER TWO SEASONS DURING 2005-2007. .. 91
FIGURE 4.2 TOTAL NUMBER OF LEAFHOPPER SPECIES COLLECTED OVER TWO
GROWING SEASONS AT FOUR SITES IN THE OVENS VALLEY REGION ,
NORTHEAST VICTORIA , USING THREE SAMPLING METHODS (PAN TRAP , STICKY
TRAP AND SWEEP NET ). OVERLAPPING AREAS INDICATE THE NUMBER OF
LEAFHOPPER SPECIES COMMON FOR EACH TRAP TYPE ...... 96
FIGURE 4.3 SEASONAL ACTIVITY AND ABUNDANCE OF SIX SELECTED LEAFHOPPER
SPECIES COLLECTED OVER TWO SEASONS , DURING 2005-2007 IN THE OVENS
VALLEY REGION OF NORTHEAST VICTORIA . POOLED DATA IS SHOWN FROM
FOUR FIELD SITES FOR WEEKLY SAMPLE DATES ...... 98
FIGURE 5.1 COMPARISON OF CATCHES OF OROSIUS ORIENTALIS USING YELLOW
STICKY TRAPS AND YELLOW PAN TRAPS FROM SEPTEMBER 2006 TO FEBRUARY
2007 FROM FIELD SITES A-D [LSD = 3.64, * P = 0,05]...... 120
FIGURE 5.2 POPULATION DYNAMICS OF OROSIUS ORIENTALIS OVER TWO SEASONS
USING THREE TRAPPING METHODS AT FIELD SITES A-D...... 121
FIGURE 6.1 OVIPOSITION CHAMBER FOR OROSIUS ORIENTALIS ON (A) HOST -PLANT
WITH (B) RUBBER BAND CLOSURE , (C) FABRIC MESH VENTILATION POINT , (D)
PETRI DISH , (E) ACCESS POINTS FOR NYMPH REMOVAL AND ADULT ADDITION,
FOAM PLUGS TO PREVENT INSECT ESCAPE AND (F) LEAF ATTACHED TO WHOLE
BEAN OR CELERY HOST -PLANT ...... 135
FIGURE 6.2 FEEDING CHAMBER FOR REARING OROSIUS ORIENTALIS ON LIQUID DIET
THROUGH A DOUBLE LAYER OF PARAFILM M...... 139
FIGURE 6.3 THE EFFECT OF TWO ARTIFICIAL DIET FORMULATIONS , MED-1 AND PT-
07, ON THE SURVIVAL AND DEVELOPMENT OF FIRST INSTARS OF OROSIUS
ORIENTALIS . EACH DATA POINT REPRESENTS THE MEAN OF 10 REPLICATES ,
XIII EACH OF WHICH CONTAINED 5 INSECTS AT THE COMMENCEMENT OF THE
EXPERIMENT ...... 143
FIGURE 6.4 THE EFFECT OF GALANTHUS NIVALIS AGGLUTININ (GNA), WHEAT GERM
AGGLUTININ (WGA) AND COWPEA TRYPSIN INHIBITOR (C PTI) WHEN
INCORPORATED AT 0.1% (WT /VOL ) IN ARTIFICIAL DIET PT-07 ON THE
SURVIVAL OF FIRST INSTAR NYMPHS OF OROSIUS ORIENTALIS . EACH DATA
POINT REPRESENTS THE MEAN OF TEN REPLICATES , EACH OF WHICH CONTAINED
FIVE INSECTS AT THE COMMENCEMENT OF THE EXPERIMENT ...... 145
FIGURE 7.1 CHAMBER TO STUDY THE FEEDING BEHAVIOUR OF OROSIUS ORIENTALIS
ON AN ARTIFICIAL DIET. ONE ELECTRODE IS CONNECTED TO THE LEAFHOPPER
WHILE THE OTHER IS PLACED IN THE DIET THROUGH A SIDE -OPENING WHICH IS
SEALED POST ELECTRODE INSERTION TO PREVENT DIET LEAKAGE ...... 162
FIGURE 7.2 VISUAL REPRESENTATION OF THE DISTINCTIVE ELECTRICAL
PENETRATION GRAPH WAVEFORMS PRODUCED BY ADULT OROSIUS ORIENTALIS
FEEDING ON BEAN (O1-O5), TOBACCO (O1-O4) AND AN ARTIFICIAL DIET (O1-
O3); PD = POTENTIAL DROP ...... 164
FIGURE 7.3 MEAN TIME TAKEN FOR OROSIUS ORIENTALIS TO REACH WAVEFORMS
O1-O5 WHEN PLACED ON AN ARTIFICIAL DIET , TOBACCO PLANTS AND BEAN
PLANTS ...... 168
FIGURE 7.4 MEAN NUMBER OF FEEDING WAVEFORMS (O1-O5) AND NON -PROBING
(NP ) PHASES OCCURRING FROM OROSIUS ORIENTALIS FEEDING ON AN
ARTIFICIAL DIET , TOBACCO PLANTS AND BEAN PLANTS ...... 169
FIGURE 7.5 MEAN DURATION OF WAVEFORMS (O1-O5) AND NON -PROBING (NP )
PHASES OCCURRING FROM OROSIUS ORIENTALIS FEEDING ON AN ARTIFICIAL
DIET , TOBACCO PLANTS AND BEAN PLANTS ...... 170
XIV LIST OF TABLES
TABLE 2.1 . LIST OF PREFERRED , TRANSIENT AND UNCONFIRMED HOST -PLANTS FOR
OROSIUS ORIENTALIS ...... 21
TABLE 3.1 LEAFHOPPERS COLLECTED FROM SEVEN FIELD SITES (A-G) DURING THE
THREE SURVEY SEASONS FROM 2005-2008...... 60
TABLE 3.2 PREVALENCE OF TBYDV IN SAMPLES OF OROSIUS ORIENTALIS
COLLECTED FROM DIFFERENT FIELD SITES DURING 2005-2008 ...... 62
TABLE 3.3 AVERAGE MINIMUM AND MAXIMUM TEMPERATURE AND TOTAL ANNUAL
RAINFALL RECORDED AT FIELD SITES A-D FROM OVER THREE YEARS (2005-07) ...... 65
TABLE 3.4 PLANT SPECIES TESTED FOR PRESENCE OF TBYDV OBTAINED FROM
FOUR FIELD SITES (A, B, C AND D) COLLECTED DURING THREE GROWING
SEASONS ...... 66
TABLE 4.1 DIVERSITY AND ABUNDANCE OF LEAFHOPPER SPECIES RECORDED USING
THREE SAMPLING METHODS (PAN TRAP , STICKY TRAP AND SWEEP NET ) AT FOUR
FIELD SITES IN THE OVENS VALLEY , NORTHEAST VICTORIA DURING TWO
COLLECTION SEASONS DURING 2005-2007...... 92
TABLE 5.1 NUMBER OF OROSIUS ORIENTALIS RECORDED FROM TWO SEASONS
(05/06 AND 06/07) AND FOUR SITES (A, B, C AND D) USING THREE DIFFERENT
TRAPPING METHODS (SWEEP NET , STICKY TRAP AND PAN TRAP )...... 117
TABLE 5.2 AVERAGE MINIMUM AND MAXIMUM TEMPERATURE AND TOTAL ANNUAL
RAINFALL RECORDED AT THE FOUR FIELD SITES (A–D) OVER 3 YEARS ...... 118 1 TABLE 6.1 COMPOSITION (MG /L) OF ARTIFICIAL DIETS , PT-07 AND MED-1 , USED
FOR REARING OROSIUS ORIENTALIS...... 137
TABLE 7.1 MAJOR CHARACTERISTICS OF THE WAVEFORMS RECORDED USING A DC
EPG SYSTEM FOR ADULT OROSIUS ORIENTALIS FEEDING ON BEANS
(WAVEFORMS O1-O5), TOBACCO (O1-O4) OR AN ARTIFICIAL DIET (O1-O3)...... 165
XV LIST OF ABBREVIATIONS
°C degrees Celsius µL microlitre µm micrometre AC alternating current AUD Australian dollar BCTV Beet curly top virus BGMV Bean golden mosaic virus BPH rice brown planthopper cm centimetre COPD chronic obstructive pulmonary disease CpTi cowpea trypsin inhibitor CR common region CTAB cetyltrimethylammonium bromide DC direct current dicot dicotyledonous DNA deoxyribonucleic acid dNTP deoxynucleotide triphosphate DPI Department of Primary Industries e extracellular e.g. exempli gratia emf electromotive force EPG electrical penetration graph g gram GLH rice green leafhopper GNA Galanthus nivalis agglutinin ha hectares Hz Hertz i intracellular IPM integrated pest management IR intergenic region
XVI kb kilobase KOH potassium hydroxide L. Linnaeus L:D light:dark LIR large intergenic regions LSD Least Significant Difference m metre M molar masl metres above sea level mg milligram min minutes mL millilitre mm millimetres mM millimolar monocots monocotyledonous plants MP movement protein mRNA messenger ribonucleic acid MSV Maize streak virus mV millivolt n number NE North-East no. number np non-probing NSP nuclear shuttle protein NSW New South Wales ori origin of replication PCR polymerase chain reaction PD Pierce’s disease pd potential drop pers. comm personal communication pers. observ. personal observation R resistance Ra variable resistor
XVII ref. reference REn replication enhancer Ri input resistor s seconds SE South-East SIR small intergenic region ss-/ds-DNA single-stranded/double-stranded deoxyribonucleic acid sscDNA single-stranded circular DNA SW South-West TbYDV Tobacco yellow dwarf virus TrAP transactivator protein TYD Tobacco yellow dwarf TYDVF/TYDVR TbYDV-specific primers TYLCV Tomato yellow leaf curl virus unpubl. unpublished USA United States of America V volt v/v volume per volume Vs voltage source WGA wheat germ agglutinin wt/vol weight per volume Ω Ohm
XVIII STATEMENT OF ORIGINAL AUTHORSHIP
The work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other higher education institution. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made.
Signature:……………………………… Date:……….18-12-09………………
XIX ACKNOWLEDGEMENTS
There are many people who have had an enormous impact on this work and on my personal development and to whom I wish to thank. Firstly, my supervisors, especially Dr Kevin Powell from Department of Primary Industries (DPI Rutherglen) for the great opportunity, mentoring and expert guidance and insight into insect physiology and without whom this project would not be possible. He provided a great working environment and always found time no matter what. My sincere thanks goes to my principal supervisor Associate Professor Rob Harding (QUT) for his advice, support and guidance through the molecular/virus world. I thank him for long inspiring conversations radiating with energy and enthusiasm in research that motivated me. Special thanks to Dr Brendan Rodoni (DPI, Knoxfield) for his time, valuable help, encouragement and powerful source of inspiration and energy.
Very special thanks goes to team from DPI Ovens especially Gary Baxter for all the help and expert advice, Jack Harrington, Anna Harrington, Guiseppe Sacko for assisting collecting samples. I wish to thank farmers of the Ovens Valley for allowing access to their properties.
I would like to thank also insect feeding behaviour expert Dr Freddy Tjallingii; leafhopper taxonomists Dr Murray Fletcher and Linda Semeraro, virologist Dr Fiona Constable and Bonny van Rijswijk. DPI Rutherglen colleagues especially, Dr Kim Andrews, Ginger Korosi, Dr Dario Stefanelli, Carolyn Trethowan. Biometricians, Sorn Norng and Dr Subhash Chandra and Jane Moran for encouragement and supporting this project.
A special thanks to QUT panel members Professor Adrian Herington, Dr Jason Geijskes and A/Prof. Anthony Clarke.
I am very grateful to Dr Angela Freeman and Dr Jo Luck for supporting my PhD studies while working at DPI Horsham.
XX
The generous support from Horticulture Australia Limited and the Tobacco Research and Development Corporation with in-kind support from the Department of Primary Industries, Victoria and QUT is greatly appreciated.
Chcialbym szczególnie podziekowac moim rodzicom i rodzenstwu w Polsce and a big thanks to my family and friends in Australia.
Finally I would like to thank my wife Kelly and our boys Aleksander and Dominik, without your encouragement and support this would not be possible.
Thank you
XXI
CHAPTER 1
AIMS AND OBJECTIVES
Description of scientific problem investigated
Tobacco yellow dwarf virus (TbYDV, genus Mastrevirus , family Geminiviridae ) is indigenous to Australia and causes the economically significant summer death and yellow dwarf diseases in bean ( Phaseolus vulgaris L.) and tobacco ( Nicotiana tabacum L.), respectively. Prior to the commencement of this project, TbYDV was known to be transmitted by the leafhopper, Orosius orientalis Evans (Hemiptera, Cicadellidae) although uncertainty remained over other potential leafhopper vectors. Further, outbreaks of the diseases caused by TbYDV were sporadic and the life-cycle of the leafhopper vector, O. orientalis , on alternate weed hosts, and subsequent acquisition and transmission periods for TbYDV, was poorly understood. Although the vector is predominantly controlled using regular sprays of systemic insecticides, the timing, frequency and efficiency of chemical applications has not been determined and/or optimised. This PhD project was instigated in an attempt to improve the basic understanding of the tritrophic associations between the vector, the virus and its host-plants, and was a collaborative industry-focused research project developed between the Queensland University of Technology (QUT) and the Department of Primary Industries (DPI), Rutherglen, Victoria. At the commencement of this project, tobacco was the most important crop in the region with the highest incidence and yield losses to TbYDV. As such, the majority of the research for this PhD was focussed on tobacco. Some bean farms were also examined, however, since beans were considered a potential alternative crop when commercial tobacco production ceased in Australia in 2007.
Overall objectives of the study
There is a substantial lack of knowledge regarding the epidemiology of many plant viruses and the tritrophic interactions that exist between viruses, insect vector/s and host-plant(s). This lack of fundamental knowledge renders
1 management of the vectors and pathogens difficult and often ineffective. The overall objective of this project was to improve the understanding of the epidemiology of TbYDV, its host-plant and vector range as well as characterizing the population dynamics and physiology of the main insect vector of TbYDV. It was envisaged that an understanding of these interactions would provide an opportunity to improve management practices by allowing reduced and optimised insecticide use, introduction of less toxic control options and the use of novel protective agents to reduce the incidence and impact of the vector and associated diseases.
Specific aims of the study
The specific aims of this study were to: (i) characterise the plant host range of both TbYDV and its primary insect vector, (ii) further investigate the insect vector(s) of TbYDV, (iii) optimise the sampling protocol for leafhopper vectors in an agricultural environment, (iv) better understand the epidemiology of TbYDV by examining vector population dynamics, (v) develop an artificial diet system for the primary insect vector of TbYDV as a bioassay for rapid screening of potential control agents, (vi) conduct a preliminary screening of potential novel anti- metabolic compounds for control of the primary insect vector and (vii) understand and characterise the feeding behaviour of the primary vector in planta and in vitro using susceptible plant hosts, non-host-plants and an artificial diet to improve our understanding of the mechanisms of TbYDV transmission.
Account of scientific progress linking the scientific papers
The three published and one accepted scientific papers derived from this PhD project are contained in Chapters 3-6. Chapter 7 describes work that is currently being prepared for publication but which has not yet been submitted.
The first paper (Chapter 3) focused on the identification and characterization of host-plants and insect vectors of TbYDV and provides insights into the epidemiology of bean summer death and tobacco yellow dwarf diseases. From extensive field-based surveys conducted in the Ovens Valley North East Victoria, three major host-plants ( Amaranthus retroflexus , Raphanus raphanistrum and Phaseolus vulgaris ), one non-preferred host-plant ( N. tabacum ) and one primary
2 leafhopper vector ( O. orientalis ) were identified. By comparing a number of field sites with different climatic conditions and different disease pressures, a number of factors were identified which could play a major role in occurrence of tobacco yellow dwarf and bean summer death diseases.
In Chapter 4, the seasonal activity and diversity of a range of possible leafhopper vectors of TbYDV and other plant pathogens in the Ovens Valley was investigated. In this paper, 57 described leafhopper species (including many economically important pests) and 15 undescribed species were identified in and around the field borders of commercially grown tobacco crops. Twenty-three species identified had not been previously recorded in Victoria. The chapter also described a number of trapping methods that are recommended for leafhoppers and compared the efficiency of the traps and leafhopper population dynamics, abundance and species diversity between sites.
Chapter 5 focused on the major leafhopper vector of TbYDV, O. orientalis . This paper described the evaluation of three sampling methods for monitoring the insect, namely sweep net, yellow pan trap and yellow sticky trap, and discussed the merits of each trap type. The seasonal activity and population dynamics of O. orientalis was examined and found to be characterised by a trimodal peak. The distribution of the leafhopper at the four sites along the Ovens Valley was also examined.
Chapters 6 and 7 describe research undertaken to provide a basic understanding of the feeding physiology, nutritional requirements and feeding behaviour of the primary vector of TbYDV, O. orientalis .
Chapter 6 described the development of first chemically defined artificial diet and feeding system for O. orientalis . This novel artificial diet formulation allowed survival of O. orientalis for up to 46 days which included full development from first instar through to adulthood. Additionally, three plant-derived proteins, cowpea trypsin inhibitor (CpTi), Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA) were assessed for antimetabolic activity by examining leafhopper survival and development. GNA and WGA were found to reduce O.
3 orientalis survival and development and thus represent potentially useful antimetabolic agents for future control strategies.
Chapter 7 described the adoption and utilisation of an electrophysiological technique (electrical penetration graph, EPG) for the first time to study the feeding behaviour of adult O. orientalis on a preferred host-plant ( P. vulgaris ), a non-host-plant ( N. tabacum ) and a simple artificial diet. This paper described the identification, analysis and characterisation of the five most prevalent EPG waveforms (O1-O5) that represent different feeding activity. The differences in duration and occurrence of the waveforms on plants and the artificial diet was discussed and related to feeding behaviour on the different feeding sources and discussed in terms of virus acquisition and transmission.
4 CHAPTER 2
LITERATURE REVIEW
The Australian tobacco industry
Tobacco in Australia – a historical perspective Tobacco (Nicotiana tabacum L) was introduced into Australia around 1850 when migrant gold-miners brought seeds into the country and grew tobacco for their private consumption. By 1889, the tobacco cropping area had reached 2.687 ha with NSW leading production with 1,955 ha followed by Victoria and Queensland with 681 and 49 ha, respectively. In 1989, North Queensland had become the major tobacco-producing district in Australia with 2,672 ha producing 7,747,320 kg of flue-cured tobacco, whilst Victoria cropped 1,830 ha and NSW 269 ha (Tonello & Gilbert 1990). Over the past 15 years, however, the number of growers declined rapidly particularly in Queensland due to a government-funded structural adjustment scheme. As a result, from 2002, the Ovens and Murray regions in Victoria led tobacco production in Australia with over 100 commercial growers until 2007 when tobacco production ceased in Australia. From 2007 onwards growers began evaluating several alternative crop options including field beans ( Phaseolus vulgaris L.) which are now widely grown and gaining popularity in the Ovens and Murray regions in Victoria (G. Baxter pers. comm.).
Cultivated tobacco Nicotiana tabacum , is a member of the Solanaceae (nightshade) family which is indigenous to North and South America. Apart from tobacco, the family includes many plants that are important sources of human food including tomato, potato, capsicum and aubergine. Tobacco plants contain high concentrations of nicotine, an alkaloid that is a strong neurotoxin. In tobacco, nicotine acts as a chemical
5 defence agent and is harmful to some insects. However, the stimulatory effects of nicotine have been exploited by humans through smoking or chewing cured tobacco leaves. Unfortunately, there are extremely harmful side-effects associated with tobacco smoking. It is highly addictive, and strongly linked to lung and other cancers, and cardiovascular and respiratory diseases such as chronic obstructive pulmonary disease (COPD) (Moszczynski 2005). Up to 2007 there were only a few varieties of tobacco grown in Victoria and Queensland with the main varieties being Dynes, Speight, K399 and RG11. The main differences between varieties were their growth rate and habit, time until the leaves “ripen”, fertiliser requirements and yield. The varieties also differ in their susceptibility to tobacco yellow dwarf disease, with Speight and K399 typically showing severe symptoms with Dynes tending to be less severely affected (Moran & Rodoni 1999).
Production systems and management The production of healthy seedling transplants greatly influences the harvested crop condition and yield. Originally, tobacco transplants were produced in the field using seedbeds (Collins & Hawks 1993). More recently, transplants are produced in greenhouses using the floating system (Smith & Boyette 1995) whereby seeds are planted directly into “cells” present in styrofoam™ trays. Trays usually contain 198 “cells” and float on shallow reservoirs containing nutrient- rich water. When the seedlings reach a height of about 10 to 12cm, they are ready for transplanting in the field. Before transplanting in the field, the soil must be reasonably moist, treated with herbicides and pesticides and ploughed. Seedlings are planted 46-56 cm apart in rows and each row can vary from 122-132 cm apart. Irrigation plays an extremely important role in tobacco production since both water stress and oversupply have considerable effects on crop quality. In Australia, the majority of farmers used irrigation systems throughout the growing season. Harvesting usually starts between 10-14 weeks from planting and lasts until all leaves are collected which can continue up to a further 10 weeks. Harvesting begins when the leaves “ripen”. Ripening starts at the base of the plant, and is characterised by a gradual change in leaf colour from green to greenish-yellow to yellow, at which
6 time the leaves are ready to be harvested (Tonello & Gilbert 1990). Tobacco production is very labour intensive with most aspects of production and management done by hand. The tobacco plant is susceptible to many pests and diseases (Lucas 1975). As a consequence, many farmers tend to overuse a variety of different chemicals in an attempt to gain maximum crop yields. Unfortunately, many of these chemicals have a detrimental effect on the environment and there is great risk of destroying fragile ecosystems and reducing water quality.
Major diseases of tobacco Tobacco production is affected by a variety of different diseases caused by a range of pathogens including fungi, bacteria, phytoplasmas and viruses. These can substantially decrease yield and quality and cause economic losses. They have also resulted in the overuse of chemicals which has a negative impact on the farming system. Blue mold, which is caused by the fungus Peronospora tabacina Adam , can be a severe plant bed disease which occurs during extended wet weather. Other important fungal diseases of tobacco include black root rot, damping-off and black shank (Lucas 1975). Bacteria can cause infections of both the foliage and roots. The main bacterial diseases include Granville wilt, hollow stalk and black leg, frenching, crown gall and leafy gall (Lucas 1975). Tobacco production is also affected by a number of different viruses. Some examples of virus diseases include tobacco yellow dwarf, tobacco mosaic, tomato spotted wilt, alfalfa mosaic and tobacco vein mottle. Of these, tobacco yellow dwarf disease, caused by Tobacco yellow dwarf virus (TbYDV), is considered the most economically important in Australia.
7 Tobacco yellow dwarf disease
Introduction The first visual record of tobacco yellow dwarf disease (TYD) in Australia dates back to 1929 when Dickson (1929) observed that, in tobacco growing areas of Myrtleford, Victoria, “dwarfing of plants is quite common in certain fields”. Several years later, McDonald (1936) reported up to 38% of “stunted” plants in experimental trials at Myrtleford. Hill (1937) was the first to actually describe the disease and propose the name “yellow dwarf”. Hill defined TYD as follows: “diseased plants usually have as many leaves as adjoining healthy ones, but the stem is only one-third the normal length, the internodes begin very short. The leaves are proportionately small, of poor quality and generally unsuitable for commercial requirements. The flowers and capsules are few and in other respects appear normal. Diseased plants flower at about the same time as healthy ones, but the leaves mature earlier, those on the lower third of the plant often being dead before the seed begins to ripen. The root system does not differ in general characteristic from that of healthy plants, but it is less extensive, the roots appearing slightly brown externally and in the region of the phloem”. He also suggested that an insect was responsible for transmission and reported that the disease was transmissible by grafting and budding (Hill 1937; Thomas & Bowyer 1979). Later studies suggested that the leafhopper Thamnotettix argentata (Evans) (Hemiptera, Cicadellidae), now known as Orosius orientalis (Matsumura), was the main vector of TYD (Day et al. 1952; Day & McKinnon 1951; Helson 1942; 1950; Hill 1941; Thomas & Bowyer 1979; van Rijswijk et al. 2002). The disease has been observed in all mainland states in Australia (Hill & Helson 1949). TYD disease was considered to be the main disease that occurs in tobacco in the Ovens and King Valleys in Victoria. Infected tobacco plants could be found on an annual basis. Surveys conducted in Victoria in the 1991/92 growing season showed that 35% to 45% of crops were infected with up to 80% infection in individual paddocks. Financial losses due to TYD infection in the 1998/99 season have been reported to be as high as $4 million AUD (Moran & Rodoni 1999).
8 Disease symptoms can appear at all growth stages, although young plants soon after transplanting appear most susceptible and show the most characteristic symptoms. In young infected plants, symptoms include folding under of leaf tips, interveinal chlorosis with a proportional reduction in height and the development of a bronzed-yellowish colour in the leaves (Figure 2.1) (Hill & Mandryk 1954). Yield losses are greatest in “early yellow dwarf plants” which remain stunted and do not produce leaves of any marketable quality. Further, the chemical characteristics of infected leaves might be different to healthy leaves.
9 10
Figure 2.1 Healthy (left) and TYD-affected tobacco plants (right) in the field in the Ovens Valley.
11
12 Etiology A viral etiology for tobacco yellow dwarf (TYD) was first suggested by Thomas (1979) who observed geminate particles in purified extracts from infected plants. Morris et al. (1992) subsequently cloned and sequenced the genome of the virus, called TbYDV, and generated an infectious clone which caused typical disease symptoms when inoculated onto tobacco plants (Morris et al. 1992).
Tobacco yellow dwarf virus TbYDV belongs to the genus Mastrevirus , family Geminiviridae . Geminiviruses are an important family of plant pathogens that have been known in tropical and subtropical regions since the mid-1800s (Wege et al. 2000), but were first described in the late 1970s (Goodman 1977a,b). They are characterised by geminate particles which contain a genome of single-stranded circular DNA (sscDNA) molecules of about 2.5–3 kb (Goodman 1981; Lazarowitz 1992; Timmermans et al. 1994). Geminviruses are classified into four genera based on genome structure, host range and insect vector (Rojas et al. 2005). In terms of insect transmission and host range, mastreviruses mainly infect monocotyledonous plants (monocots) and are leafhopper transmitted, curtoviruses infect dicotyledonous plants (dicots) and are leafhopper transmitted, topocuvirus infect dicots and are treehopper transmitted and begomoviruses infect dicots and are whitefly transmitted. The differences in the genome structure of mastreviruses, curtoviruses and begomoviruses are outlined in Figure 2.2. The topocuvirus genome structure (not shown) is a hybrid of the mastrevirus and monopartite begomovirus genomes.
13
Figure 2.2 Genome organization of the three main geminivirus genera. (A, B, C) begomovirus, (D) curtovirus, (E) mastrevirus (Source: Rojas et al . 2005).
14 The begomovirus genome is either bipartite or monopartite (Figure 2.2A). The bipartite begomovirus genome, represented by the type member Bean golden mosaic virus (BGMV) and is composed of two ~2.6 kb DNA molecules, designated DNA-A and DNA-B. The only sequence similarity is a ~200 bp region referred to as the common region (CR) that contains the viral origin of replication (ori), with a characteristic stem-loop structure containing the invariant nonanucleotide sequence, TAATATTAC, which functions as the initiation site for rolling circle replication. Genes are named according to the DNA component, and the DNA strand on which they are encoded [viral (V) or complementary (C)- sense], and the relative proximity to the CR; alternatively, genes are named according to their function (CP, capsid protein; Rep, replication-associated protein; TrAP, transactivator protein; REn, replication enhancer; NSP, nuclear shuttle protein; and MP, movement protein). The monopartite begomovirus genome (Figure 2.2B) is represented by Tomato yellow leaf curl virus (TYLCV) and is composed of a single ~3 kb DNA molecule similar to the DNA-A of the bipartite members, except that it has an additional V1, or MP, gene. The ori is contained in the intergenic region (IR). Most Old World monopartite begomoviruses (Figure 2.2C) are associated with a ~1.4 kb satellite DNA, referred to as DNA-β. The curtovirus genome (Figure 2.2D), is represented by the type member Beet curly top virus (BCTV) and is composed of a single ~3 kb DNA with an IR containing the ori. The complementary-sense genes are similar in organization to those of the monopartite begomoviruses, whereas V2 and V3 are involved in modulation of ss-/ds-DNA levels and movement, respectively. The mastrevirus genome (Figure 2.2E), is represented by the type member Maize streak virus (MSV), and is composed of a single ~2.7 kb DNA and has both large and small intergenic regions, LIR and SIR, respectively. Mastreviruses express two Reps: Rep, expressed via a spliced mRNA from the C1 and C2 ORFs; and RepA, expressed from a C1-derived mRNA. RepA interacts with the host retinoblastoma-related protein (Rojas et al . 2005). Although the majority of mastreviruses infect monocots, TbYDV infects a dicot, tobacco. The complete genome of a TbYDV isolate from a tobacco plant growing in the Ovens Valley, Victoria, has been sequenced and comprises 2.58 kb (Morris et al. 1992). Based on this sequence, van Rijswijk et al . (2004) designed a
15 pair of TbYDV-specific primers (TYDVF and TYDVR) to amplify a 509 bp fragment of the movement protein-coding region. These primers were subsequently used in our epidemiological study to detect TbYDV reservoirs and vectors.
The vector of TbYDV – Orosius orientalis
Vector taxonomy Orosius orientalis , known also as the common brown leafhopper, has been proposed as a main vector of TbYDV (Helson 1942, 1950; Hill 1941; Hill & Helson 1949) but was only confirmed using molecular techniques in an unpublished report (van Rijswijk et al. 2002). Orosius orientalis belongs to the large and diverse non-endopterygote order of Hemiptera and is further classified into the Auchenorrhyncha suborder and Cicadellidae (leafhoppers) family (Gullan & Cranston 2005; Triplehorn & Johnson 2005). In addition to leafhoppers, the Auchenorrhyncha also contains cicadas (Cicadidae), spittlebugs (Cercopoidae), treehoppers (Membracoidae) and planthoppers (Fulgoroidea). All leafhoppers are herbivorous, sap-sucking insects which feed using a needle-like stylet that contains two canals, one to deliver saliva and a second for fluid uptake. The adult size ranges from a few millimetres up to 30 mm. Leafhoppers can communicate with each other acoustically in a similar way to cicadas, by creating songs using specific sound-producing organs or tymbals that are located on the end of abdomen.
Vector characteristics Orosius orientalis (Figure 2.3) was initially classified as Thamnotettix argentata (Evans) and O. argentatu s (Evans). Despite the economical importance and wide distribution of O. orientalis , taxonomically it has been known under a number of different names (Ghauri 1966). The adult is described as follows: “Length of female: 3.2 to 3.5 mm; length of male: 2.9 to 3.0 mm; Head: pale yellow, marked with an irregular dark-brown pattern; eyes: dark-brown; Pronotum: anterior third
16 pale yellow, posterior two-thirds grey, flecked with transverse dark-brown markings; Scutellum: yellow, but for the apex, which is dark brown; Tegmen: hyaline, with a silvery appearance, due to the sheen of the underlying wings, patterned with an irregular network of dark-brown markings; Thorax and abdomen ventral surface: pale yellow with scattered dark-brown markings” (Evans 1938). The insect is not native to Australia but is widely distributed across the continent and occurs in many central Pacific islands, the Philippines and Malaysia. It is thought to have been originally introduced into Australia from India (Ghauri 1966).
Life stages of Orosius orientalis Oviposition commences when the adult female makes an incision, usually in the stem, petiole or leaf mid-rib, and lays a single egg in a slit-like chamber. Eggs are on average 0.8 mm in length and 0.3 mm wide. Initially the egg appears translucent but, with embryonic development, it progressively becomes cream and eyes develop to become visible as two dark spots. Five nymphal instars have been characterised. The newly hatched nymph is 1.8 mm in length (Helson 1942). Wing-pads are visible in the third instar and the fully grown fifth instar is 2.8 to 3.4 mm long. Early instars, once hatched stay in close proximity to the oviposition site and usually feed on the basal surface of the leaf. If disturbed, nymphs can hop short distances. Under controlled environments, the egg incubation period varies from 7 to 22 days (Helson 1942). In laboratory conditions, the adult male can live for 125 days and the adult female for 240 days. In both laboratory and field, three generations have been observed per season (Helson 1942).
Host-plants and population dynamics Adult and nymphal stages of leafhoppers feed on plant phloem (Day et al. 1952) and usually do not move unless disturbed. They can feed on a wide range of host- plants and can survive on less preferable hosts for some time. There are a number of different host-plants reported for O. orientalis (Table 2.1) (Helson 1942). On some plants, eggs hatch but nymphs fail to mature - these are regarded as transient hosts. In contrast, preferred hosts will support O. orientalis through their life-
17 cycle. Orosius orientalis can feed on tobacco when there is no other source of food available but it does not breed on this plant (Helson 1942). Research into the population dynamics of leafhoppers in the tobacco- growing region of the Ovens Valley over a single season only has shown that common brown leafhoppers are most abundant from the end of November until early January (Helson, 1942). Although attempts were made to determine which environmental conditions influenced population dynamics, the results were inconclusive.
18
Figure 2.3 Orosius orientalis , the common brown leafhopper.
19
20 Table 2.1 . List of preferred, transient and unconfirmed host-plants for Orosius orientalis
Host status Family Species Common name Ref.
Preferred host Apiaceae Apium graveolens Celery h Asteraceae Callistephus chinensis Aster d,b, h Cryptostemma calendulaceae Cape weed a, c Hypochaeris radicata Cat's ear a,b,d Boraginaceae Heliotropium europeum Common heliotrope a Sisymbrium orientale Wild mustard a Chenopodiaceae Beta vulgaris Beet a,c,d Chenopodium carinatum Keeled goosefoot a Fabaceae Arachis hypogaea Peanut i Medicago denticulata Bur clover a,b,c Phaseolus vulgaris French bean g Trifolium repens White clover a,b,c Trifolium subterraneum Clare subclover a,f Geraniaceae Erodium cicutarium Common crowfoot a,c,d Erodium cygnorum Blue crowfoot a,b,d
21 Malvaceae Malva parviflora Cheeseweed a,f Modiola caroliniana Small-flowered mallow a,b Pittosporaceae Bursaria spinosa Blackthorn a,c Portulacaceae Portulaca oleraceae Pigweed a Solanaceae Datura stramonium Common thornapple a,c,h,j Solanum laciniatum Kangaroo thornapple a Solanum melongena Egg plant a Solanum nigrum Nightshade a Transient Chenopodiaceae Chenopodium alba Fat hen a Cucurbitaceae Cucumis myriocarpus Paddy melon a Fabaceae Chamaecytisus palmensis Tree lucerne a Medicago sativa Lucerne a,b,h Lamiaceae Marrubium vulgare Horehound a Myrtaceae Eucalyptus rostrata River red gum a Poaceae Hordeum sp. Barley a Paspalum dialatatum Paspalum a Secale cereale Rye a
22 Polygonaceae Polygonum aviculare Wireweed a Solanaceae Lycopersicon esculentum Tomato a,d,h Nicotiana glauca Tree tobacco a Nicotiana suaveolens Australian tobacco a Nicotiana tabacum Tobacco a,d Solanum tuberosum Potato a,e Undetermined Amaranthaceae Amaranthus retroflexus Redroot Pigweed m Apiaceae Daucus carota Carrot f Apocynaceae Vinca rosea Periwinkle d Asteraceae Silybum marianum Variegated thistle b,d Sonchus oleraceus Common sow thistle b,d Brassicaceae Brassica adpressa Hairy brassica b,d Raphanus raphanistrum Wild Radish m Caricaceae Carica papaya Papaya k Chenopodiaceae Atriplex semibaccata Creeping saltbush d Convolvulaceae Cuscuta australis Dodder g Fabaceae Cicer arietinum Chickpea i
23 Crotalaria intermedia Slenderleaf crotalaria f Crotalaria juncea Sunn hemp i Desmodium canum Creeping beggarweed f Desmodium uncinatum Desmodium silverleaf f Glycine max Soybean i Lotononis bainesii Miles Lotononis f Stylosanthes species Stylo f Trifolium pratense Red clover f Vigna radiate Mung bean i Vigna unguiculata Cowpea i Loranthaceae Amyema miquelii Box mistletoe n Plantaginaceae Plantago lanceolatum Ribwort d Solanaceae Nicotiana glutinosa Wild tobacco h,j Vitaceae Vitis vinifera European grapevine l a (Helson 1942), b (Hill & Helson 1949), c (Helson 1949), d (Helson 1950), e (Norris 1954), f (Hutton & Grylls 1956), g (Bowyer & Atherton 1971), h (Bowyer 1974), i (Iwaki et al. 1978), j (Harding & Teakle 1985), k (Padovan & Gibb 2001), l (Beanland 2002), m (Tr ębicki et al. 2010), n (Burns 2009).
24 Alternative vectors of TbYDV Initial investigations into alternate vectors of TbYDV were conducted in the early 1950s and were based entirely on symptomology, which is at best insensitive and at worst unreliable. Examination and comparisons of the stylet-feeding tracts caused by the known vector, O. orientalis, and other species of leafhoppers (Day et al. 1952) failed to identify any potential alternative vectors. Using sensitive PCR-based techniques, TbYDV was detected in three additional leafhoppers Balclutha spp., Anzygina spp. and Limotettix incertus (van Rijswijk et al. 2002 , 2004). However, the presence of virus in these insects does not necessarily mean that they are vectors, since the insects may have recently fed on a virus-infected plant or the samples may have been contaminated with minute amounts of TbYDV-infected plant material. As such, acquisition and transmission studies need to be carried out, in combination with molecular techniques, to conclusively determine whether the insects are vectors.
Control strategies for TbYDV
Despite the importance of yellow dwarf disease, the epidemiology of TbYDV is poorly understood and there is no effective control for the disease. The current approaches used to control the disease normally involve the use of insecticides and herbicides. However, because of the undesirable environmental effects, alternative approaches are being sought that can improve farming practice and achieve better results with minimum chemical input (Tr ębicki et al . 2007).
Vector and disease management Integrated pest management (IPM) is a concept and strategy that combines different practices in pest and disease control. Disease management is an important production practice in tobacco. Optimum disease control is best achieved by implementing all available methods in an IPM program. In agriculture, the different methods to control diseases include cultural control such as production of transplants and crop rotation. Other control options include breeding for host-plant resistance, biological control, optimising chemical
25 management and the use of transgenic plants (Shew & Lucas 1991). Although the latter approach is arguably the most effective method and tobacco is indeed one of the more readily modified crop species, the commercial growing of genetically modified tobacco is banned in Australia.
Crop rotation Crop rotation is the practice of growing different types of plants in the same field over sequential seasons to reduce accumulation of pests and diseases by reducing the availability of host-plants on which vectors feed and subsequently acquire and transmit pathogens. Crop rotation also offers a chance to balance soil nutrients like nitrogen, phosphorus and potassium content and improve soil structure and fertility. Furthermore, rotation of the crop may also be very important for reducing weeds, many of which are hosts for many insect vectors and viruses. As such, crop rotation should be considered as a very important tool in pest and disease management (Shew & Lucas 1991).
Leafhopper management Leafhopper management in the field is a complex task. The only form of management of O. orientalis currently used is to reduce the number of insects and minimise the possibility of virus transmission by either application of insecticides to control the insect directly or herbicides to reduce availability of feeding sites on a regular basis. The best reported strategy to control the leafhopper, and therefore reduce spread of TbYDV, is with three or six weekly insecticidal sprays (Paddick et al. 1971). Since mass migration of the vector from the main host to the tobacco crop is thought to occur only a few times during the season (Helson 1942), regular spraying can reduce the incidence of tobacco yellow dwarf disease. Although this strategy has proven somewhat successful, the approach is difficult to justify in terms of both cost and detrimental environmental impacts (Hill & Mandryk 1954). To be cost-effective, a much better understanding of the disease epidemiology is required so that new, more effective insecticides can be identified and their timing and rate of application be optimised. Such information can be best obtained
26 through the development and use of artificial insect diet system in which novel chemicals can be screened as well as through feeding behaviour studies.
Leafhopper artificial diets
Leafhoppers can survive on a wide range of host-plants, which can make it difficult to determine their essential nutritional requirements and model their population dynamics. One proven way to determine their nutritional requirements is through the use of an artificial diet system where dietary components can be manipulated and optimised to suit the individual leafhopper species (Cohen 2004).
Leafhopper nutrition There are many potential benefits that can be derived from rearing insects on artificial diet feeding systems. Using an artificial diet, it is possible to (i) better understand an insects nutritional requirements and characterise its feeding behaviour, (ii) screen a wide range of conventional and novel control agents and (iii) modify diet components to determine what would be the impact of a management change (eg. manipulating water or nutrient inputs) on the insect. A considerable amount of research has been done on artificial diet for sap-sucking insects, and insects reared on artificial diets are used in many different research programs (Cohen 2004) including the development of biological control agents and sterile insect technologies (Knipling 1979), the use of bioreactors for pharmaceutical productions (Hughes & Wood 1998), and the provision of food sources for different animals (Versoi & French 1992). Although no artificial diet has yet been developed for O. orientalis , artificial diets have been developed for several Hemiptera and other insect orders (Vanderzant 1974). There are a number of essential dietary components for all insects including amino acids, carbohydrates, cholesterol, inorganic salts and vitamins (Chapman 1982; Vanderzant 1974). Because Hemiptera are essentially sap-sucking fluid feeders, a feeding delivery system for a liquid diet is essential. The first published record of this was delivering a liquid diet through a thin membrane (Carter 1927) to feed sugar beet leafhopper, Eutettix tenellus (Baker),
27 (Figure 2.4). Mittler and Dadd (1962) provided a breakthrough in rearing homopterous insects on artificial diets by using a liquid synthetic diet and thin Parafilm™ membrane. They successfully reared the aphid Myzus persicae (Sulzer) through successive generations; the same method was found to be suitable for leafhoppers and planthoppers (Koyama & Mitsuhashi 1969). Attempts to rear sap-sucking insects on an artificial diet have however met with limited success. In most cases, the insect survival rates have been very low due to the inadequacy of both the membrane and dietary components (Mitsuhashi 1974) and insects do not always reach adulthood on the diet suggesting a sub-optimal diet or feeding system has been developed. The most significant results in the rearing of leafhoppers and planthoppers on artificial diets were reported by Mitsuhashi and Koyama (Koyama 1979, 1981; Mitsuhashi 1974, 1979; Mitsuhashi & Koyama 1972, 1975, 1977). They were able to rear rice planthoppers and leafhoppers on an artificial diet successfully and suggested that survival rates were higher than those achieved with insects reared on the host-plant (Koyama et al. 1981). Recently, research has focussed on developing specific diets for aphids, planthoppers and leafhoppers to enable the identification of possible toxic anti-metabolic agents which might be used as insect control agents (Powell et al. 1993, 1995; Powell 2001). Using artificial diets to rear rice and taro planthoppers, Nilaparvata lugens (Stål) and Tarophagous proserpina (Kirkaldy), respectively and the rice leafhopper Nephotettix cinciteps (Uhler), the lectins Galanthus nivalis agglutinin (GNA) and concanavalin A were shown to exert significant anti-metabolic effects (Powell et al. 1995 1998, 2001).
28
Figure 2.4 An example of an artificial diet feeding chamber for sap-sucking insects (Source: Powell et al . 1993).
29 Leafhopper feeding behaviour
Electrophysiological techniques The feeding physiology of O. orientalis is poorly understood largely due to technological difficulties. For many years, these same problems hampered attempts to study the feeding physiology of Hemiptera. A breakthrough in this area of research came when a method of electronically recording aphid feeding behaviour, later known as electrical penetration graph was developed (McLean & Kinsley 1964). The EPG technique was later improved (Tjallingii 1978) and has subsequently undergone many modifications. The main change to the original AC (alternating current) EPG system was the use of DC (direct current) EPG (Tjallingii 1978). The basic principals of both AC and DC EPG are the same; they measure changes in electrical resistance in the plant and stylet probing insect. To measure the changes, the insect is attached or tethered (using electrically conductive paint) to an electrode by means of thin gold or platinum wire (Figure 2.5, Figure 2.6). The tethered insect is then transferred to the plant and an output electrode is placed in the soil or plant. Upon feeding, the insects’ stylet penetrates the plant tissue, a circuit is closed and an electric signal can be amplified, recorded and analysed. The signals can produce different waveforms depending on insect feeding activity (Tjallingii 1988) and the different patterns of voltage fluctuation can be correlated with insect activity and stylet location within the plant tissue. Correlation of different waveforms with insect activity and stylet location can be achieved using a variety of techniques including histology, simultaneous video recording, honeydew production, artificial diet observations, plant hydrostatic pressure characteristics and radioactive labelling (Walker 2000).
30
A.
B. Figure 2.5 . The components of an EPG monitoring system. A - Components of the primary circuit that are housed within the EPG monitor. B – Diagram of the primary circuit where the biological components are modelled as a variable resistor, Ra. Other symbols: Ri, input resistor (fixed resistor); Vs, voltage source (Source: Walker 2000)
31 EPG is a very important tool used to study the feeding behaviour of many families of Hemiptera. Since first developed, it has been mainly used to study insects in the family Sternorrhyncha, especially aphids (McLean & Kinsey 1964; McLean & Kinsey 1965; Prado & Tjallingii 1994; Tjallingii 1978, 1988; Tjallingii & Hogen Esch 1993), and to a lesser extent whiteflies (Walker & Janssen 2000) and phylloxera (Harrewijn et al. 1998; Kingston et al. 2005; Kingston 2007). Relatively few EPG studies have been published on leafhoppers and planthoppers (Khan & Saxena 1988; Kimmins 1989; Powell & Gatehouse 1996; Seo et al. 2009) and no studies have been done using O. orientalis or other suspected vectors of TbYDV. In recent years using the AC EPG system, a considerable amount of research on the stylet activities of the glassy-winged sharpshooter, Homalodisca coagulata (Say) (Hemiptera: Cicadellidae: Cicadellinae), the vector of Pierce’s disease (PD) of grapevine, has been published to provide an improved understanding of the processes involved with leafhopper stylet penetration (Backus 2005; Joost et al. 2006). Using the DC EPG, a few leafhopper species were studied recently and a number of waveforms have been correlated to specific feeding activities (Kimmins & Bosque-Perez 1996; Lett et al. 2001; Miranda et al. 2009; Stafford & Walker 2009).
32
Figure 2.6 Orosius orientalis attached via silver conductive glue to a gold wire to an EPG insect electrode (not shown) and feeding on a Phaseolus vulgaris leaf.
33
34 Host-plant selection Apart from aphids, little research has been done to fully determine how Hemiptera select their host-plant (Backus 1988). Research on some aphid species has shown that long distant orientation depends mainly upon visual colour cues (Kennedy 1966; 1976; Kennedy & Ludlow 1974) and to a lesser extent upon olfactory cues (Chapman et al. 1981). Research done on the leafhopper Amrasca devastans (Distant) suggests that the insect uses olfactory cues to differentiate between host and non-host-plant (Saxena & Saxena 1975). The rice leafhopper ( Nephotettix virescens Distant) expresses different feeding behaviour in the presence of neem oil (Saxena & Khan 1986). In addition, the brown rice planthopper ( N. lugens ) reacts to non-hosts plant after being sprayed with volatile rice-plant chemicals (Saxena & Khan 1986).
Feeding strategies There are two behavioural strategies engaged by Hemiptera during feeding; sheath feeding and lacerate-flush feeding (Miles 1972). Sheath feeding is more complex than lacerate-flush feeding and is characterised by sealing the insect stylet tips into a vascular cell via a salivary sheath. In this strategy, the insect uses two types of salivary secretions: a watery, digestive enzyme saliva and a rapidly solidifying (lipoproteinaceous) or gel saliva which is secreted while the insect is feeding in the plant tissue (Miles 1968). In lacerate flush feeding the insect produces watery saliva that contains digestive enzymes (Miles 1978). In this feeding process the stylet moves very rapidly in the plant tissue and lacerates mesophyll and parenchymal cells in the process (Smith & Poos 1931). In contrast to sheath feeding, insects using the lacerate-flush strategy do not produce a salivary sheath and are primarily mesophyll feeders (Miles 1968).
Summary
TbYDV is indigenous to Australia and causes the economically significant summer death and yellow dwarf diseases in bean and tobacco, respectively. Prior to the commencement of this project, TbYDV was known to be transmitted by the
35 leafhopper, O. orientalis although uncertainty remained over other potential leafhopper vectors. Further, outbreaks of the diseases caused by TbYDV were sporadic and the life cycle of the leafhopper vector, O. orientalis , on alternate weed hosts, and subsequent acquisition and transmission periods for TbYDV, was poorly understood. Although the vector is predominantly controlled using regular sprays of systemic insecticides, the timing, frequency and efficiency of chemical applications has not been determined and/or optimised. This PhD project was instigated in an attempt to improve the basic understanding of the tritrophic associations between the vector, the virus and its host-plants, and was a collaborative industry-focused research project developed between the Queensland University of Technology (QUT) and the Department of Primary Industries (DPI), Rutherglen, Victoria.
References
Backus EA. 1988. Sensory systems and behaviours which mediate hemipteran plant-feeding: a taxonomy overview. Journal of Insect Physiology 34 , 151-165.
Backus EA. 2005. Stylet penetration by adult Homalodisca coagulata on grape: Electrical penetration graph waveform characterization, tissue correlation, and possible implications for transmission of Xylella fastidiosa . Annals of the Entomological Society of America 98 , 787-813.
Beanland L. 2002. Developing Australian grapevine yellows management strategies. GWRDC Final Report DNR00/02, Melbourne, Victoria, Australia, 1- 24.
Bowyer JW. 1974. Tomato big bud, Legume little leaf, and Lucerne witches' broom: three diseases associated with different mycoplasma-like organisms in Australia. Australian Journal of Agricultural Research 25 , 449-457.
36 Bowyer JW & Atherton JG. 1971. Summer death of French bean: new hosts of the pathogen, vector relationship and evidence against mycoplasmal etiology. Phytopathology 61 , 1451-1455.
Burns AE. 2009. Diversity and dynamics of the arthropod assemblages inhabiting mistletoe in remnant eucalypt woodlands. Charles Sturt University, New South Wales, Australia. PhD Thesis.
Carter W. 1927. A technique for use with homopterous vectors of plant diseases, with special reference to the sugar beet leafhopper, Eutettix tenellus (Baker). Journal of Agricultural Research 34 , 449-451.
Chapman RF. 1982. Chapter V. Nutrition. In: Insects: Structure and Function. Hodder and Staughton, Kent, UK. 88-99.
Chapman RF, Bernays EA & Simpson SJ. 1981. Attraction and repulsion of aphid, Cavariella aegopodii , by plant foods. Journal of Chemical Ecology 7, 881- 889.
Cohen AC. 2004. Insect Diets. Science and Technology, CRC Press LLC, Boca Raton, USA.
Collins WK & Hawks JSN. 1993. Principles of Flue-cured Tobacco. North Carolina State University, USA.
Day MF, Irzykiewicz H & McKinnon A. 1952. Observations on the feeding of the virus vector Orosius argentatus (Evans), and comparisons with certain other jassids. Australian Journal of Scientific Research, Series B - Biological Sciences 5, 128-142.
Day MF & McKinnon A. 1951. A study of some aspects of the feeding of the jassid Orosius . Australian Journal of Scientific Research, Series B - Biological Sciences 4, 125-135.
Dickson BT. 1929. The work of the Division of Economical Botany, for the year 1928-29. Journal of the Council for Scientific and Industrial Research 14 , 19-22.
37 Evans, JW. 1938. Australian leafhoppers (Jassoidea, Homoptera). Part 8. Papers and Proceedings of the Royal Society of Tasmania 1938, 1-18
Ghauri MSK. 1966. Revision of the genus Orosius Distant (Homoptera: Cicadelloidea). Bulletin of the British Museum (Natural History), Entomology 18 , 231-252.
Goodman RM. 1977a. A new kind of virus is discovered. Illinois Research 19, 5.
Goodman RM. 1977b. Single stranded-DNA genome in a whitefly-transmitted plant virus. Virology 83 , 171 - 179.
Goodman RM. 1981. Geminiviruses. Journal of General Virology 54 , 9-21.
Gullan PJ & Cranston PS. 2005. The Insect. An Outline of Entomology. Blackwell Publishing, Oxon, UK.
Harding RM & Teakle DS. 1985. Mycoplasma-like organisms as causal agents of potato purple top wilt in Queensland. Australian Journal of Agricultural Research 36 , 443-449.
Harrewijn P, Piron PGM & Ponsen MB. 1998. Evolution of vascular feeding in aphids: an electrophysiological study. Proceedings of Experimental and Applied Entomology 9, 29-34.
Helson GAH. 1942. The leafhopper Thamnotettix argentata Evans, a vector of tobacco yellow dwarf. Journal of Council of Scientific and Industrial Research 15 , 175-184.
Helson GAH. 1950. Yellow dwarf of tobacco in Australia V. Transmission by Orosius argentatus (Evans) to some alternative host-plants. Australian Journal of Agricultural Research 1, 144-147.
Hill AV. 1937. Yellow dwarf of tobacco in Australia, I. Symptoms. Journal of Council of Scientific and Industrial Research 10 , 228-230.
38 Hill AV. 1941. Yellow dwarf of tobacco in Australia, II. Transmission by the jassid Thamnotettix argentata (Evans). Journal of the Council for Scientific and Industrial Research 14 , 181-186.
Hill AV & Helson GA. 1949. Distribution in Australia of three virus diseases and of their common vector Orosius argentatus (Evans). Journal of the Australian Institute of Agricultural Science 15 , 160-161.
Hill AV & Mandryk M. 1954. A study of virus diseases "big bud" of tomato and "yellow dwarf" of tobacco. Australian Journal of Agricultural Research 5, 617- 625.
Hughes PR & Wood HA. 1998. Production of pharmaceuticals and other recombinant proteins in insect larvae. SIM News 48 , 100-105.
Hutton EM & Grylls NE. 1956. Legume 'little leaf', a virus disease of subtropical pasture species. Australian Journal of Agricultural Research 7, 85-97.
Iwaki M, Roechan M, Seleh N, Sugiura M & Hibino H. 1978. Identity of mycoplasma-like agents of legume witches' brooms in Indonesia. Contributions Central Research Institute for Agriculture 41 , 1-11.
Joost PH, Backus EA, Morgan D & Yan F. 2006. Correlation of stylet activities by the glassy-winged sharpshooter, Homalodisca coagulata (Say), with electrical penetration graph (EPG) waveforms. Journal of Insect Physiology 52 , 327-337.
Kennedy JS. 1966. The balance between antagonistic induction and depression of flight activity in Aphis fabae Scopoli. Journal of Experimental Biology 45 , 215- 228.
Kennedy JS. 1976. Host-plant finding by flying aphids. Symposium Biologica Hungarica 16 , 121-123.
Kennedy JS & Ludlow AR. 1974. Co-ordination of two kinds of flight activity in an aphid. Journal of Experimental Biology 61 , 173-196.
39 Khan ZR & Saxena RC. 1988. Probing behaviour of three biotypes of Nilaparvata lugens (Homoptera: Delphacidae) on different resistant and susceptible rice varieties. Journal of Economic Entomology 81 , 1338-1345.
Kimmins FM. 1989. Electrical penetration graphs from Nilaparvata lugens on resistant and susceptible rice varieties. Entomologia Experimentalis et Applicata 50 , 69-79.
Kimmins FM & Bosque-Perez NA. 1996. Electrical penetration graphs from Cicadulina spp. and the inoculation of a persistent virus into maize. Entomologia Experimentalis et Applicata 80 , 46-49.
Kingston K, Powell KS & Cooper P. 2005. Investigating the digestive function and feeding behaviour of grape phylloxera. Abstracts of Comparative Biochemistry and Physiology 141A , 116.
Kingston KB. 2007. Digestive and feeding physiology of grape phylloxera (Daktulosphaira vitifoliae Fitch), Australian National University, Canberra, Australian Capitol Territory, Australia. PhD thesis.
Knipling EF. 1979. The basic principals of insect population suppression and management. USDA Agriculture Handbooks 512 . 659 pp.
Koyama K. 1979. Rearing of the brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae) on synthetic diet. Japanese Journal of Applied Entomology and Zoology 23 , 39-40.
Koyama K. 1981. Survival of the larvae of the brown planthopper Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) on sugar solutions. Japanese Journal of Applied Entomology and Zoology (In Japanese) 25 , 125-126.
Koyama K & Mitsuhashi J. 1969. Artificial feeding of small brown planthopper, Laodelphax striatellus Fallen (Hemiptera: Delphacidae). Japanese Journal of Applied Entomology and Zoology 13 , 89-90.
40 Koyama K, Mitsuhashi J & Nasu S. 1981. Rearing of the planthopper, Sogatella longifurcifera Esaki et Ishihara (Hemiptera: Delphacidae) on synthetic diet. Japanese Journal of Applied Entomology and Zoology (In Japanese) 25 , 198-200.
Lazarowitz SG. 1992. Geminiviruses: genome structure and gene function. In: CRC Critical Reviews in Plant Sciences , pp. 327-349, (Ed, BV Conger). CRC Press, Bocca Raton, Florida, USA.
Lett J-M, Granier M, Grondin M, Turpin P, Molinaro F, Chiroleu F, Peterschmitt M & Reynaud B. 2001. Electrical penetration graphs from Cicadulina mbila on maize, the fine structure of its stylet pathways and consequences for virus transmission efficiency. Entomologia Experimentalis et Applicata 101 , 93-109.
Lucas GB. 1975. Diseases of Tobacco , 3rd Ed. Biological Consulting Associates, Raleigh, North Carolina, USA.
McDonald WJB. 1936. Tobacco investigation in Victoria. Results of 1935 test. Journal of the Department of Agriculture of Victoria , 34 217-225.
McLean DL & Kinsey MG. 1964. A technique for electronically recording aphids feeding and salivation. Nature (London) 202 1358-1359.
McLean DL & Kinsey MG. 1965. Identification of electrically recorded curve patterns associated with aphid salivation and ingestion. Nature 205 , 1130-1131.
Miles PW. 1968. Insect secretions in plants. Annual Review of Phytopathology 6, 137-164.
Miles PW. 1972. The saliva of Hemiptera. Advances in Insect Physiology 9, 183- 255.
Miles PW. 1978. Redox reactions of hemipterous saliva in plant tissue. Entomologia Experimentalis et Applicata 24 , 534-539.
Miranda MP, Fereres A, Appezzato-da-Gloria B & Lopes JRS. 2009. Characterization of electrical penetration graphs of Bucephalogonia xanthophis , a vector of Xylella fastidiosa in citrus. Entomologia Experimentalis et Applicata 130 , 35-46.
41 Mitsuhashi J. 1974. Methods for rearing leafhoppers and planthoppers on artificial diets. Review of Plant Protection Research 7, 57-67.
Mitsuhashi J. 1979. Chapter 10: Artificial rearing and aseptic rearing of leafhopper vectors: Applications in virus and MLO research. In: Leafhopper Vectors and Plant Disease Agents. (Eds K Maramorosch & KF Harris). Academic Press, New York, USA, 369-412.
Mitsuhashi J & Koyama K. 1972. Artificial rearing of the smaller brown planthopper, Laodelphax striatellus Fallén, with special reference to rearing conditions for the first instar nymphs. Japanese Journal of Applied Entomology and Zoology 16 , 8-17.
Mitsuhashi J & Koyama K. 1975. Oviposition of the smaller brown planthopper, Laodelphax striatellus , (Hemiptera: Delphacidae) into various carbohydrate solutions through a parafilm membrane. Applied Entomology and Zoology 10 , 123-129.
Mitsuhashi J & Koyama K. 1977. Effects of amino acids on the oviposition of the smaller brown planthopper, Laodelphax striatellus , (Hemiptera: Delphacidae). Entomologia Experimentalis et Applicata 22 , 156-169.
Mittler TE & Dadd RH. 1962. Artificial feeding and rearing of the aphid Myzus persicae (Sulzer) on a completely defined synthetic diet. Nature 195 , 404.
Moran JR & Rodoni B. 1999. Strategies for the control of tobacco yellow dwarf virus. Report on a consultancy conducted on behalf of the Tobacco Corporation of Victoria. Knoxfield, Victoria, Australia. 1-5.
Morris BAM, Richardson KA, Haley A, Zhan X & Thomas JE. 1992. The nucleotide sequence of the infectious cloned DNA component of Tobacco yellow dwarf virus reveals features of geminiviruses infecting monocotyledonous plants. Virology 187 , 633-642.
Moszczynski P. 2005. Health harms have induced by cigarette smoking. Przeglad Lekarski 62 , 253-256.
42 Norris DO. 1954. Purple-top wilt, a disease of potato caused by tomato big-bud virus. Australian Journal of Agricultural Research 5, 1-11.
Paddick RG, French FL & Turner PL. 1971. Control of leafhopper-borne plant disease possibly due to direct action of systemic biocides on mycoplasmas. Plant Disease Reporter 55 , 291-293.
Padovan AC & Gibb KS. 2001. Epidemiology of phytoplasma diseases in papaya in Northern Australia. Journal of Phytopathology 149 , 649-658.
Powell K. 2001. Antimetabolic effects of plant lectins towards nymphal stages of the planthoppers Tarophagous proserpina and Nilaparvata lugens . Entomologia Experimentalis et Applicata 99 , 71-77.
Powell KS, Gatehouse AMR, Hilder VA & Gatehouse JA. 1993. Antimetabolic effects of plant lectins and plant and fungal enzymes on the nymphal stages of two important rice pests, Nilaparvata lugens and Nephotettix cinciteps . Entomologia Experimentalis et Applicata 66 , 119-126.
Powell KS, Gatehouse AMR, Hilder VA, Van Damme EJM, Peumans WJ, Boonjawat J, Horsham K & Gatehouse JA. 1995. Different antimetabolic effects of related lectins towards nymphal stages of Nilaparvata lugens . Entomologia Experimentalis et Applicata 75 , 61-65.
Powell KS & Gatehouse JA. 1996. Mechanism of mannose-binding snowdrop lectin for use against brown planthopper in rice. In: Rice Genetics III. Proceedings of the Third International Rice Genetics Symposium (Ed GS Khush) 753-758. IRRI, Manila, Philippines.
Powell KS, Spence J, Bharathi M, Gatehouse JA & Gatehouse AMR. 1998. Immunohistochemical and developmental studies to elucidate the mechanism of action of the snowdrop lectin on the rice brown planthopper, Nilaparvata lugens (Stål). Journal of Insect Physiology 44 , 529-539.
Prado E & Tjallingii WF. 1994. Aphid activities during sieve element punctures. Entomologia Experimentalis et Applicata 72 , 157–165.
43 Rojas MR, Hagen C, Lucas WJ & Gilbertson RL. 2005. Exploiting chinks in the plant's armor: evolution and emergence of geminiviruses. Annual Review of Phytopathology 43 , 361-394.
Saxena KN & Saxena RC. 1975. Patterns of relationship between certain leafhoppers and plants. Part III. Range and interaction of sensory stimuli. Entomologia Experimentalis et Applicata 18 , 194-206.
Saxena RC & Khan ZR. 1986. Aberrations caused by neem oil odour in green leafhopper feeding on rice plants. Entomologia Experimentalis et Applicata 42 , 279-284.
Seo BY, Kwon Y-H, Jung JK & Kim G-H. 2009. Electrical penetration graphic waveforms in relation to the actual positions of the stylet tips of Nilaparvata lugens in rice tissue. Journal of Asia-Pacific Entomology 12 , 89-95.
Shew HD & Lucas GB. 1991. Compendium of Tobacco Diseases. The Disease Compendium Series of the American Phytopathological Society . APS, St. Paul, Minnesota, USA.
Smith DF & Boyette M. 1995. Transplant production. North Carolina Cooperative Extension Bulletin 16-29.
Smith FF & Poos FW. 1931. The feeding habits of some leafhoppers of genus Empoasca . Journal of Agricultural Research 43 , 267-285.
Stafford CA & Walker GP. 2009. Characterization and correlation of DC electrical penetration graph waveforms with feeding behaviour of beet leafhopper, Circulifer tenellus . Entomologia Experimentalis et Applicata 130 , 113–129.
Thomas JE. 1979. Tobacco yellow dwarf and bean summer death viruses. University of Sydney, New South Wales, Australia. PhD Thesis,
Thomas JE & Bowyer JW. 1979. The purification and identification of tobacco yellow dwarf and Bean summer death viruses. Australasian Plant Pathology 8, 36-38.
44 Timmermans MCP, Das OP & Messing J. 1994. Geminiviruses and their uses as extrachromosomal replicons. Annual Review of Plant Physiology and Plant Molecular Biology 45 , 79-112.
Tjallingii WF. 1978. Electronic recording of penetration behaviour by aphids. Entomologia Experimentalis et Applicata 24 , 721-730.
Tjallingii WF. 1988. Electrical recording of stylet penetration activities. In: Aphids, their Biology, natural Enemies and Control (Eds AK Minks & P Harrewijn) 95-108. Elsevier, Amsterdam,The Netherlands.
Tjallingii WF & Hogen Esch TH. 1993. Fine structure of aphid stylet routes in plant tissue in correlation with EPG signals. Physiological Entomology 18 , 317- 328.
Tonello PEA & Gilbert EJ. 1990. Tobacco growing in Queensland. Queensland Department of Primary Industries, Regional Series RQM90002, Queensland, Australia.
Tr ębicki P, Powell KS & Baxter G. 2007. Epidemiology and management of leafhopper that transmit a unique virus In: Sustainable tobacco production. (Eds Baxter G, Hayes G, Harrington A, Harrington J & Sacco G.) Department of Primary Industries, Final Report, TBO4001, 132, Rutherglen, Victoria, Australia.
Tr ębicki P, Harding RM, Rodoni B, Baxter G & Powell KS. 2010. Vectors and alternative hosts of TbYDV in south-eastern Australia. Annals of Applied Biology 157 , 13-24 .
Triplehorn CA & Johnson NF. 2005. Borror and Delong's Introduction to the Study of Insects. Thomson Book/Cole, USA. van Rijswijk B, Rodoni BC, Baxter G & Moran JR. 2002. Managing tobacco yellow dwarf virus. Phase 1: Developing a diagnostic tool. Phase 2: Beginning to understand the epidemiology of TYDV. In: Tobacco Research and Development Corporation Final Report 1999/2001 of Project DAV 35T, Department of Natural Resources and Environment, Knoxfield, Victoria, Australia. pp.48.
45 van Rijswijk B, Rodoni BC, Revill PA, Thomas JE, Moran JR & Harding RM. 2004. Analysis of variability in partial sequences of genomes of Tobacco yellow dwarf virus isolates. Australasian Plant Pathology 33 , 367-370.
Vanderzant ES. 1974. Development, significance and application of artificial diets for insects. Annual Review of Entomology 19 , 139-160.
Versoi PL & French LK. 1992. The establishment of commercial insectaries. In Advances in Insect Rearing for Research and Pest Management . (Eds TE Anderson & NC Leppa). Westview Press, New Delhi, 457-463.
Walker GP. 2000. A beginner's guide to electronic monitoring of Homopteran probing behavior, pp. 14-40. In: Principles and Applications of Electronic Monitoring and Other Techniques in the Study of Homopteran Feeding Behaviour (Eds GP Walker & EA Backus) Entomological Society of America, Lanham, MD, USA.
Walker GP & Janssen JAM. 2000. Electronic recording of Whitefly (Homoptera: Aleyrodidae) feeding and oviposition behaviour. In: Principles and Applications of Electronic Monitoring and Other Techniques in the Study of Homopteran Feeding Behaviour (Eds GP Walker & EA Backus) pp. 172-200. Entomological Society of America, Lanham, MD, USA.
Wege C, Gotthardt RD, Frischmuth T & Jeske H. 2000. Fulfilling Koch’s postulates for Abutilon mosaic virus. Archives of Virology 145 , 2217-2225.
46 CHAPTER 3
VECTORS AND ALTERNATIVE HOSTS OF TbYDV IN SOUTH-EASTERN AUSTRALIA
Piotr Tr ębicki 1, 2 , Rob M. Harding 1, Brendan Rodoni 3, Gary Baxter 4
& Kevin S. Powell 2
1Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Queensland, GPO Box 2434, Brisbane, 4001, Australia. 2Department of Primary Industries, Biosciences Research Division, RMB 1145, Chiltern Valley Road, Rutherglen, Victoria, 3685, Australia. 3Department of Primary Industries, Private Bag 15, Ferntree Gully Delivery Centre, Knoxfield, Victoria, 3180, Australia. 4TAFCO Rural Supplies, Great Alpine Highway, Myrtleford, Victoria, 3737, Australia.
Annals of Applied Biology (2010), 157 , 13-24
47
STATEMENT OF AUTHORSHIP
Piotr Tr ębicki (principal author): Executed the work (collected samples, designed and conducted all laboratory experiments, analysed and interpreted data) and wrote initial manuscript.
Signed ……………………………………. Date.. 18-12-09……………………..
Rob Harding: Conceived project idea, critically interpreted data and contributed to final manuscript.
Signed ……………………………………. Date.. 18-12-09…….………………..
Brendan Rodoni: Conceived project idea, critically interpreted data and contributed to final manuscript.
Signed ……………………………………. Date 18-12-09…...…………………..
Gary Baxter: Conceived project idea, critically interpreted data and contributed to final manuscript.
Signed ……………………………………. Date …18-12-09..…………………..
Kevin Powell: Conceived project idea, supervised the work, critically interpreted data and contributed to final manuscript.
Signed ……………………………………. Date …18-12-09...…………………..
48 Abstract
Factors that determine the epidemiology of Tobacco yellow dwarf virus (TbYDV), including alternative host-plants and insect vector(s), were assessed over three consecutive growing seasons at four field sites in Northeastern Victoria in commercial tobacco growing properties. In addition these factors were assessed for one growing season at three bean growing properties. Overall twenty-three leafhopper species were identified at the seven sites, with Orosius orientalis as the predominant leafhopper. Of the leafhoppers collected, only O. orientalis and Anzygina zealandica tested positive for TbYDV by PCR. The population dynamics of O. orientalis was assessed using sweep net sampling over three growing seasons and a trimodal distribution was observed. Despite large numbers of O. orientalis occurring early in the growing season (September-October), TbYDV was only detected in these leafhoppers between late November and the end of January. The peaks in the detection of TbYDV in O. orientalis correlated with the observation of disease symptoms in tobacco and bean and were associated with warmer temperatures and lower rainfall. Spatial and temporal distribution of vegetation at selected sites was determined using quadrat sampling. Of 40 plant species identified, TbYDV was detected in only four dicotyledonous species, Amaranthus retroflexus , Phaseolus vulgaris , Nicotiana tabacum and Raphanus raphanistrum . The proportion of host and non-host availability for leafhoppers was associated with climatic conditions.
49 Introduction
Tobacco yellow dwarf virus (TbYDV, family Geminiviridae, genus Mastrevirus ) is an important pathogen in Australia causing summer death and yellow dwarf disease in bean (Phaseolus vulgaris L.) and tobacco ( Nicotiana tabacum L.), respectively (Hill, 1941; Helson, 1950; Thomas & Bowyer, 1979; Thomas & Bowyer, 1980). Infection can result in annual losses of up to four million Australian dollars in tobacco (Tr ębicki et al . 2007) and symptoms of TbYDV infection and associated crop losses have been observed in bean crops in the Ovens Valley region (Tr ębicki pers. observ.) TbYDV is a leafhopper-transmitted, phloem-restricted mastrevirus which is indigenous to Australia. Although most mastreviruses infect monocotyledonous plants, TbYDV together with Bean yellow dwarf virus from South Africa and Chickpea chlorotic dwarf Pakistan virus from Pakistan (Nahid et al. 2008), infect dicotyledonous plants. The complete genomic sequence of an Australian TbYDV isolate, derived from a tobacco plant growing in the Ovens Valley, Victoria, has been characterised and was shown to be monopartite and comprised of 2.58 kb of single-stranded, circular DNA (Morris et al. 1992). The majority of studies investigating the epidemiology of TbYDV were conducted in the 1940’s when specific and sensitive molecular diagnostic tests were unavailable and disease diagnosis was based on host-plant symptoms. Hill (1941) and later Helson (1950) reported that the leafhopper Orosius orientalis (Matsumura) (Hemiptera: Cicadellidae), previously described as O. argentatus (Evans) and Thamnotettix argentata (Evans), was a vector of the disease, but there has been little research aimed at identifying other potential vectors. Furthermore, although numerous host-plants for TbYDV have been reported (Helson, 1950; Hill, 1950; Hill & Mandryk, 1954; Bowyer & Atherton, 1971), most of these have been identified under experimental conditions by the development of typical yellow dwarf symptoms in tobacco (Hill, 1937) following transmission from suspected host-plants by either grafting or by the vector O. orientalis . Recently, a TbYDV-specific PCR test was developed (van Rijswijk et al. 2004) which was used to determine the variability within the genome of Australian TbYDV
50 isolates. This PCR test was also used to identify the weed Raphanus raphanistrum as a host-plant of TbYDV (van Rijswijk et al . 2004). Due to the lack of detailed epidemiological data for TbYDV, disease management strategies continue to rely heavily on repeated insecticide applications which are largely ineffective and environmentally hazardous (Paddick et al. 1971; Paddick & French 1972; Osmelak 1986). The aim of this 3- year study was to use the molecular-based diagnostic test developed by van Rijswijk et al. (2004) to conduct a more thorough investigation into the epidemiology of TbYDV through seasonal monitoring of plants and Hemipteran insects in vegetative areas surrounding bean and tobacco crops at commercial farms in south-eastern Australia. Such data should assist in the development of improved management strategies to control diseases caused by TbYDV in commercial crops.
Materials and methods
Sampling sites The collection of potential leafhopper vectors and monitoring of their population dynamics was conducted over three consecutive growing seasons, 2005/06, 2006/07 and 2007/08, at four commercial tobacco ( Nicotiana tabacum L.) farms located in the Ovens Valley, north-east Victoria, Australia (Figure 3.1). These farms have been under continuous tobacco cultivation using conventional management conditions for decades. Sites A and B were located in the Lower Ovens Valley near Whorouly (36°29'23S, 146°35'48E) with an elevation of 210 metres above sea level (masl). Site C was located between Ovens and Eurobin (36°37'12S, 146°48'31E) at 246 masl while Site D was located in the Upper Ovens Valley (36°43'25S, 146°52'30E) with an elevation of 320 masl. During the 2007/08 growing season, sampling was also carried out at an additional three sites (E, F and G) near Myrtleford (36°33'S, 146°34'E) where tobacco had been previously grown but which had been subsequently replanted to beans ( Phaseolus vulgaris L. cv. “Borlotti”).
51 Site selection was based on the historical incidence of TbYDV symptoms in commercially grown tobacco. Historically, tobacco growing in the Lower Ovens Valley region (sites A and B) is most affected by TbYDV, with decreasing disease incidence recorded with increasing elevation (sites C and D) (G. Baxter personal observation).
Weather data Meteorological data for each collecting season was obtained from the SILO database (http://www.bom.gov.au/silo/ ). Air temperature and rainfall were recorded from three weather stations each located within five km of each experimental site.
52
Figure 3.1 Location of field sites (labelled A – G) in north-east Victoria, Australia, that were used in this study.
53 Leafhopper collection During each season, leafhoppers were collected on a weekly basis using a sweep net (38 cm diameter). Sweeping was done by swinging the net through the plant canopy at 180 ° along a 100 m transect where one metre was equivalent to one sweep (Figure 3.2). One hundred sweeps per site per sampling date were done during the 2005/06 and 2006/07 seasons, while 150 sweeps per site (sites A and B) per sampling date were done during the third season. In addition, leafhoppers were collected weekly at sites E, F and G between December 2007 and January 2008. The insects caught were immediately placed in a killing jar containing ethyl acetate for 10-15 mins before transfer into zip-lock plastic bags. To sort samples, four different laboratory test sieves were stacked on top of each other in descending pore size (aperture size: 3.35 mm; 2 mm; 1 mm and 212 µm). Samples were emptied onto the sieves and briefly shaken. Each sieve was emptied and all leafhoppers were transferred to a Petri dish for microscopic identification using taxonomic keys (Evans, 1966; Ghauri, 1966; Knight, 1975; Knight, 1987; Day & Fletcher, 1994; Fletcher, 2009 and updates). Identified specimens were transferred to a 1.5 ml test tube and stored at -80°C for DNA analysis.
54
Figure 3.2 Overview of the sampling methodology used for insects (sweep net) and plants (quadrant) at each field site.
55
56 Plant surveys To determine the potential host-plants for TbYDV, a diverse range of plant species were collected at monthly intervals at four experimental sites (A, B, C and D for the first two seasons; A and B only in the third season). In addition, plant material of selected species (namely Phaseolus vulgaris, Nicotiana tabacum, Amaranthus retroflexus and Raphanus raphanistrum ) was collected at three sites (E, F and G), on five occasions between December 2007 and January 2008. A line transect sampling method using a zigzag survey design (Strindberg & Buckland, 2004) was utilized to collect all plant material. The zig-zag survey was done both within crop and in adjacent vegetation along a 100 m transect at every site (Figure 3.2). Randomly selected specimens of each plant species located on the transect line were collected and identified to species level using field guides and classification keys (Lamp & Collet 1989; Parsons & Cuthbertson 1992; Cummins & Moerkerk 1997). Samples were placed in plastic zip-lock bags and stored at either 4°C or -80°C for further analysis using PCR to determine TbYDV presence. The quadrat survey method was used to assess the presence of potential host- plants for leafhopper vectors identified in the previous growing season (2005/06), to understand differences between the sites and to collect information regarding changes in plant temporal composition and predominant ground cover. Leafhopper host-plant surveys were carried out at monthly intervals at four locations (A-D) during one growing season only (December 2006-April 2007). Using a 1 m 2 metal quadrat, 29 measurements along 100 m long plots were taken at each sampling date from each site (Figure 3.2). To ensure the quadrat was placed in the same sampling location at each sampling date, a grid of wooden stakes was placed permanently on-site during the season at each location. For each quadrat, the plant species present, the percentage of coverage of each plant species and dry plant coverage was visually assessed. All plants were identified to species level using field guides (Lamp & Collet, 1989; Parsons & Cuthbertson, 1992; Cummins & Moerkerk, 1997). In addition to the quadrat survey in areas adjacent to the tobacco crop, during 2006 and 2007 vegetation at all seven field sites was visually assessed for symptoms of TbYDV and random symptomatic (i.e. showing yellowing and stunting) plant samples were collected and tested for presence of the virus by PCR.
57 DNA extraction and PCR DNA was extracted from leafhoppers essentially as described by Goodwin et al . (1994). Depending on the number of leafhoppers collected at each sampling point between 1-10 leafhoppers of a single species were pooled and placed in 1.5 ml Eppendorf tubes containing between 125 µl to 500 µl CTAB extraction buffer, crushed using a micropestle and incubated at 65°C for 5 min. Following a chloroform/isoamyl alcohol (24:1 v/v) extraction, the DNA was precipitated, washed twice in 70% ethanol and resuspended in nuclease-free sterile water. Samples were stored at -20°C. DNA was extracted from plants (~0.2 g of leaf midrib/petiole tissue) using a QIAGEN DNeasy® Plant Mini kit according to the protocol described by Green et al. (1999). The samples were resuspended in 100 µl TE buffer, pH 9.0 and stored at -20°C. Both insect and plant sample DNA was tested for the presence of TbYDV using the TbYDV-specific primers, TYDVF (5’ CAT TTA TAT TGG TAG GTG GAC 3’) and TYDVR (5’CCC TTA TAC CGG CCC GCC AT3’), which amplify a 509 bp product (van Rijswijk et al. 2004). Each PCR reaction (25 µl) contained 0.5 µl of DNA template, 1 µl of each primer (0.4 µmol final concentration) and either 22.5 µl of Invitrogen™ Platinum® PCR SuperMix or, a reaction master mix containing 18.75 µl of sterile (RNase-, DNase-free) water, 2.5 µl 10 x reaction buffer, 0.75 µl 50 mM MgCl2, 0.5 µl dNTP mixture (each at 10 mM). The mixture was heated at 95 °C for 10 min and subjected to 35 cycles of 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min and a final step of 72 °C for 10 min using a BioRad Icycler. Amplicons were analysed by electrophoresis through 1% TBE agarose gels and visualised by ethidium bromide staining. Three controls were included in all gels (known TbYDV infected tobacco, non-infected tobacco and no template).
Statistical analysis
Statistical analysis was performed using Genstat software (10 th Edn 2007, Lawes Agricultural Trust). A one-way analysis of variance (ANOVA) was used to compare differences between four sites (A-D) in relation to annual temperature during three years (2005-07).
58 Results
Leafhopper diversity and abundance Using a sweep net, in excess of 4300 leafhoppers were collected at the seven field sites over the three-season survey period. These leafhoppers were classified into 23 species (Table 3.1), of which two were previously undescribed. Orosius orientalis was by far the most abundant species (60%) found across all sites followed by Anzygina zealandica (Myers) (11%), previously known as Zygina zealandica (Fletcher & Larivière 2009), and Nesoclutha phryne (Kirkaldy) (10%). Relatively high numbers of Balclutha frontalis (Ferrari) (6%), Austroasca viridigrisea (Paoli) (5%), Batracomorphus angustatus (Osborn) (2%) and Arawa novella (Metcalf) (2%) were also collected. During the first two growing seasons, most O. orientalis were collected from site C (n = 1474), followed by site B (n = 625), site D (n = 422) and site A (n = 255). During the third growing season, when insects were only collected at sites A and B, the number of O. orientalis was again higher at site B (n = 452) than site A (n = 290).
Testing leafhoppers for TbYDV Of the 23 leafhopper species tested for the presence of TbYDV by PCR, a product of the expected size (~500 bp) was only amplified from O. orientalis and A. zealandica samples . Of the 313 O. orientalis samples (each containing between 1- 10 leafhoppers depending on the number collected during each sampling date) tested over the three season surveys, 70 samples (22%) were TbYDV positive (Table 3.2). Of these 70 samples, those derived from site B contained the greatest number of positives (32%) followed by site A (24%) while 10% of samples collected at sites C and D tested positive. In addition, all O. orientalis samples were positive at sites E-G Of the 83 samples containing A. zealandica that were collected, only one sample from site B, which comprised a single insect, tested positive for TbYDV.
59 Table 3.1 Leafhoppers collected from seven field sites (A-G) during the three survey seasons from 2005-2008.
Subfamily Species Common name Number collected
Agalliinae Austroagallia torrida Evans Spotted leafhopper 41 Deltocephalinae Arawa novella Metcalf 92 Chiasmus varicolor Kirkaldy 4 Exitianus plebeius Kirkaldy 21 Exitianus nanus Distant 1 Limotettix incertus Evans 21 Nesoclutha phryne Kirkaldy Australian grass leafhopper 437 Orosius orientalis Matsumura Common brown leafhopper 2579 Orosius canberrensis Evans 8 Maiestas knighti Webb & Viraktamath 10 Maiestas vetus Knight 4 Eurymelinae Balclutha frontalis Ferrari 249 Iassinae Eurymeloides pulchra Signoret 2 Typhlocybinae Batracomorphus angustatus Osborn Green jassid 95
60 Austroasca viridigrisea Paoli Vegetable leafhopper 222 Kahaono pallida Evans 8 Kahaono wallacei Evans 8 Anzygina zealandica Myers Yellow leafhopper 463 Anzygina sidnica Kirkaldy 20 Zygina evansi Ross 16 Xestocephalinae Xestocephalus australensis Kirkaldy 15
61
Table 3.2 Prevalence of TbYDV in samples of Orosius orientalis collected from different field sites during 2005-2008.
a Site Samples tested TbYDV positive samples O. orientalis A 37 9 (24%) B 148 48 (32%) C 98 10 (10%) D 30 3 (10%)
a = each sample contained 1-10 leafhoppers. Additionally Orosius orientalis was collected on a few occasions from sites E, F and G and all samples tested positive for TbYDV
62 Population dynamics of O. orientalis When the number of O. orientalis trapped at the four sites (A-D) in consecutive growing seasons was analysed, a trimodal peak was generally observed (Figure 3.3). At all sites, similar trends were observed in the seasonal activity of O. orientalis . The first population peaks occurred early in the season mid-September (early spring), the second in late November and the third in early January (summer). At sites C and D, the greatest numbers of O. orientalis were recorded in spring, whereas at sites A and B, peak numbers were recorded during the summer. The numbers of O. orientalis remained at relatively low levels from the end of January until late May. When the relative abundance of O. orientalis between sites was analysed, relatively higher numbers of leafhoppers were observed in the second survey season, a season characterised by relatively low annual rainfall and warmer temperatures (see below).
Potential alternative hosts of TbYDV A diverse range of weeds and cultivated plant species growing adjacent to commercially grown tobacco and beans were collected using a zig-zag survey and tested for the presence of TbYDV by PCR (Table 3.3). Of the 40 plant species tested, TbYDV was only detected from samples of Phaseolus vulgaris (common bean), Nicotiana tabacum (tobacco) and the weeds Amaranthus retroflexus L. and Raphanus raphanistrum L. Although both A. retroflexus L. and R. raphanistrum were identified at all sites, TbYDV-infected samples of these plant species were only detected at sites A-C and E-G. When pooled samples of each known TbYDV host-plant species (shown in Table 3), collected monthly from zig-zag surveys over the three year period, were tested using PCR, the greatest number of TbYDV-positive samples was found at site B (46%; 14 positives/30 samples), followed by site A (43%; 13/30), site C (13%; 4/30) and site D (3%; 1/30).
63
Site A Site B 300 Site C Site D
200 O. orientalis
Number of 100
0 Jul-06 Jul-07 Jan-06 Jan-07 Jan-08 Jun-06 Jun-07 Jun-08 Oct-06 Oct-06 Oct-07 Feb-06 Sep-06 Feb-07 Sep-07 Feb-08 Apr-06 Apr-07 Apr-08 Dec-05 Dec-05 Dec-06 Dec-07 Mar-06 Mar-07 Mar-08 Aug-06 Nov-06 Aug-07 Nov-07 May-06 May-06 May-07 May-08
Sample date
Figure 3.3 Population dynamics of Orosius orientalis during three growing seasons (2005/06, 2006/07 and 2007/08) from four sites (A, B, C and D) monitored using sweep netting. In the 2007-08 collecting season only sites A and B were studied.
64
Table 3.3 Average minimum and maximum temperature and total annual rainfall recorded at field sites A-D from over three years (2005-07)
Year 2005 2006 2007
Sites Temp. (°C) Rain (mm) Temp. (°C) Rain (mm) Temp. (°C) Rain (mm)
max min max. min. max. min.
A & B 21.71 7.63 815.4 23.15 7.1 333.4 22.98 8.88 653.7 C 21.32 7.82 1229.8 22.66 7.44 449.4 22.52 9.07 954.8 D 18.92 6.72 1196.8 20.22 6.47 476.8 20.04 7.86 863.5
65 Table 3.4 Plant species tested for presence of TbYDV obtained from four field sites (A, B, C and D) collected during three growing seasons
TbYDV-status of plants TbYDV-status of plants Plant species Plant species from different sites** from different sites** A B C D A B C D Acacia spp. × - - - Plantago lanceolata - × - × Amaranthus retroflexus + + + + Plantago major - - - × Bromus diandrus - - - - Polygonum aviculare × - - × Bromus mollis - - × - Polygonum hydropiper × × - × Capsella bursa-pastoris × - - × Raphanus raphanistrum + + + - Chenopodium album - - - × Rubus fruticosus × - - × Citrullus lanatus × - × × Rumex acetosella × × × - Convolvulus arvensis × - × Rumex crispus × × - × Cynodon dactylon - - × - Salvia verbenaca × - - × Cyperus egrostis × - - × Silybum marianum × - - × Datura spp .* - - - × Solanum lycopersicum - - × × Galium aparine × × - × Solanum nigrum* × - × × Humulus spp. × × - × Stachys arvensis × × - ×
66 Lactuca serriola - × - × Taraxacum officinale - - - - Lolium rigidum - - - - Trifolium repens* - - - - Malva parviflora* - - - × Trifolium subterraneum* × × - - Nicotiana tabacum + + + + Typha domingensis × × - × Olea europaea × - × × Vicia sativa × × - × Paspalum spp. - - - - Vitis spp. - - × × Phaseolus vulgaris + + × × Zea mays - × × ×
+ = PCR positive, - = PCR negative, × = plants not present at the site. *confirmed hosts plants for O. orientalis. **Phaseolus vulgaris, Raphanus raphanistrum and Amaranthus retroflexus also tested positive at sites E-
67 Using the quadrat survey, the temporal distribution and diversity of vegetation from four sites (A-D) was monitored through one season (December 2006 to April 2007) in order to assess the differences between the sites in terms of plant composition and distribution and occurrence of host-plants for the vector and the disease. Forty-two plant species were identified during this period (data not presented). Site C had the highest number of different plant species followed by sites B, A and D, respectively (Figure 3.4). Additionally, site C had the greatest diversity and density of known O. orientalis host-plant species followed by B, A and D. With the exception of site D, where the highest proportion of plant species recorded were monocotyledonous, all other sites were dominated by dicotyledonous plants. Even though monocots were represented by fewer species (sites A, B and C), they had the highest spatial density in all sites. At each site, Cynodon dactylon L. was the dominant plant species with between 15% (site B) to 40% (sites A, C and D) coverage at the beginning of the season. At sites A, B and C, Raphanus raphanistrum , a known host-plant for TbYDV, was found at 1-5 % coverage. In contrast, only single plants of another known host-plant, A. retroflexus, were recorded from all sites during the quadrat survey period. The virus status of these plants was not examined. During the survey period (December 2006 – April 2007), the neighbouring farms at sites A and B had the same total rainfall (123 mm each) which was almost two-fold lower than that received at sites C (216 mm) and D (201 mm). Despite identical rainfall at sites A and B, the proportion of senesced vegetation, dicotyledonous and monocotyledonous species at each site throughout the season was different with monocots more prevalent at site A and dicots more prevalent at site C (Figure 3.5). There was a similar trend between changes in the proportion of dry vegetation during the sampling period between sites A and D.
Seasonal detection of TbYDV Leafhoppers and plants were collected from September till the end of May over the three survey seasons on a weekly and monthly basis at four sites (A-D), respectively, and tested for the presence of TbYDV. In addition at sites E-G samples were only collected and tested once during January 2008.
68 Monitoring conducted on Cicadellidae trapped at each site indicated that, although a diverse range of leafhopper species (over 23 species) were prevalent in and around each of the commercial tobacco and bean farms, only O. orientalis was consistently trapped in high numbers in all seasons, despite relatively high numbers of leafhoppers and host-plants early in each season (during spring). No TbYDV-positive samples were detected until the end of November. TbYDV was subsequently detected from the end of November until February in O. orientalis (Figure 3.6) and four plant species ( P. vulgaris , N. tabacum , A. retroflexus and R. raphanistrum; Table 3.3). The characteristic symptoms of TbYDV were observed in both beans and tobacco between six to ten days after the O. orientalis population peak in early January 2006/07 for tobacco and early January 2008 for beans. In mid-January 2006 in a tobacco field adjacent to sites A and B around 30% of the crop was infected with TbYDV compared to site C where TbYDV infection was around 5% and site D with only a few infected tobacco plants detected around the field border. This survey was conducted based on a visual assessment of typical symptoms of TbYDV in tobacco (Hill, 1937) and confirmed by PCR. Additionally, in January 2008, up to 95% of the P. vulgaris samples collected at three bean farms (E, F and G) in Myrtleford tested positive for TbYDV by PCR and total crop failure was observed. Meteorological data collected over the three-season survey period (2005-07) showed that the average annual maximum temperatures at sites A, B and C were significantly higher (P<0.05) than that of site D (Table 3.4). Rainfall data showed that site C recorded the highest precipitation rate (apart from 2006) followed by site D and with the lowest recorded for sites A and B.
69
Figure 3.4 Proportion of total host and non-host-plants for Orosius orientalis from four field sites (A-D) recorded during 2006/07 growing season as determined by quadrat survey.
70
Figure 3.5 Changes in distribution of vegetation type [senesced (dry), dicotyledonous and monocotyledonous], rainfall and temperature during one growing season (2006/2007) across the four field sites (A, B, C and D, lines = average temperature, bars = rainfall).
71
Figure 3.6 Numbers and TbYDV status of Orosius orientalis , average temperature and rainfall during three consecutive growing seasons. Combined data from all four field sites: A, B, C and D (lines = average temperature, bars = rainfall).
72 Discussion
This is the first systematic epidemiological study of TbYDV achieved by examining the interactions between the virus, leafhopper vector/s and host-plants under field conditions. Hill (1941) first reported that O. orientalis was a possible vector of TbYDV based on the development of typical disease symptoms on healthy tobacco that had been exposed to a range of field-collected leafhoppers. In a previous study using a PCR-based diagnostic test (van Rijswijk et al. 2002), TbYDV was detected not only in Orosius orientalis , but also in the leafhoppers Balclutha spp., Limotettix incertus and Anzygina spp. In the present study up to 32% of the O. orientalis samples tested positive for TbYDV by PCR, although the relative percentage may be lower (due to sample size variation) this still provides some evidence that this insect is an important vector. This study did not detect TbYDV in Balclutha spp or L. incertus, despite regular testing over three seasons, and only one sample of 83 A. zealandica tested positive for TbYDV. Therefore it is unlikely that Balclutha spp is a vector of TbYDV as it feeds predominately on grasses (Knight 1987; Pilkington et al. 2004; Narhardiyati & Bailey 2005) and TbYDV has only been detected in dicots. Furthermore, there is no evidence that this species is a phloem feeder. In contrast L. incertus is predominantly a phloem feeder and could theoretically acquire the phloem-specific TbYDV (Day et al. 1952). However, the relatively small numbers of this leafhopper trapped in the field suggest that L. incertus is unlikely to be an important vector, at least in the area studied. Although TbYDV was detected in Anzygina spp, both in this study and that of van Rijswijk et al . (2002), this leafhopper is also considered an unlikely vector as both nymphs and adults feed preferentially on parenchymal cells (Witt & Edwards 2000) rather than phloem (Day et al. 1952). However transmission tests, using known host-plants, will ultimately be required to prove the vector status of these insects. Orosius orientalis is widely distributed in Australia, Asia and the Pacific region (Ghauri 1966). It is considered a polyphagous insect, having been recorded as feeding on over 60 plant host species, including A. retroflexus and R. raphanistrum, and confirmed to be able to breed on many of these hosts. The
73 detection of TbYDV-infected A. retroflexus and R. raphanistrum in this study supports the notion that these weeds may act as reservoirs for virus acquisition by O. orientalis . However, tobacco is not considered to act as a virus reservoir since (i) no nymphal development and only limited adult survival has been recorded on N. tabacum (Hill, 1941; Helson, 1942) and (ii) TbYDV transmission and acquisition studies have shown that O. orientalis cannot acquire the virus from TbYDV-infected tobacco (Helson, 1950). As such, tobacco should be considered a dead-end host for the virus as this plant species only becomes infected when the preferred leafhopper hosts decline and the insect is forced to feed on it. In contrast, P. vulgaris can support the insect through at least one generation and can therefore potentially serve as a virus reservoir (Bowyer & Atherton, 1971). The population dynamics of O. orientalis showed three peaks of abundance across four sites (A-D) occurring during the three survey seasons (during two tobacco growing seasons (from November-April) and one bean growing season at three sites (E-G; from December-March)). Typical symptoms of TbYDV were clearly visible in both tobacco and bean crops between six to ten days after the population peak in early January, with virus infection confirmed by PCR. This is consistent with previous studies which reported the occurrence of disease symptoms in late November, late December and early January (Hill & Allan, 1942; Hill, 1950). Interestingly, although in the present study TbYDV was detected in O. orientalis in late November, no TbYDV was detected in crop plants until January. It is possible that temperature may have a major influence on the ability of vectors to acquire and transmit TbYDV. In our study, TbYDV was detected in both insect and plants only when average temperatures were above 18°C. Research with other Cicadellid vectors has also shown that virus transmission is greatly reduced or abolished at lower temperatures (Hitoshi & Jutaro, 1980; Creamer & He 1997). A combination of many factors, including climate and vegetation, may play a major role in the incidence of TbYDV across the Ovens Valley. Clear differences were observed between sites in regards to the occurrence of disease, weed hosts and O. orientalis . Plant composition and leafhopper diversity differed between sites, especially between sites located in the Lower Valley, where the occurrence of TbYDV was highest (A and B) and sites in Upper Valley (C and D) where TbYDV was recorded relatively rarely. Sites A and B, which recorded the
74 highest incidence of TbYDV in host-plants and O. orientalis , had the lowest precipitation and the highest average temperature. Although tobacco is not a preferred host for O. orientalis , reduced primary host-plant availability at these sites, especially in mid-summer, may have encouraged the vector to feed on the irrigated tobacco crop when border vegetation had dried out. At sites C and D, which had relatively lower maximum temperature (site D) and higher rainfall than sites A and B the virus incidence was lower than that recorded from sites A and B. At site C, although host-plant species for O. orientalis were the most abundant, the incidence of TbYDV-infected host-plants bordering the field site was considerably lower than that at sites A and B. Even though the vector was in high abundance at site C, higher rainfall and less host-plants for the virus reduced the incidence of the disease. Site D was less affected by the virus because it was surrounded mainly by grazing land with a higher proportion of non-host monocotyledonous species, a very low number of suitable host-plant species for the vector and the virus, as well as a relatively high rainfall and lower temperature. Incidence of TbYDV in the vector and the host-plant appeared at the same time at each location, despite the site differences in terms of vector numbers and host-plant diversity. The results of this study have provided important information which can be used to develop more effective disease control strategies. For example, the population dynamic studies have provided data on the seasonal occurrence of O. orientalis and thus the most appropriate times for insecticide applications. Furthermore, disease occurrence in beans and tobacco could be minimised by targeting herbicide applications to TbYDV host-plants such as Amaranthus retroflexus and Raphanus raphanistrum ) that were commonly found in and around crops at the two most disease-affected sites. Apart from TbYDV, O. orientalis also transmits phytoplasmas which cause a range of economically important phytoplasma-associated diseases in Australia such as legume little leaf (Hutton & Grylls, 1956), tomato big bud (Hill & Mandryk, 1954; Osmelak, 1986), lucerne witches broom (Helson, 1951), potato purple top wilt (Grylls, 1979; Harding & Teakle, 1985) and Australian lucerne yellows (Pilkington et al . 2004). Therefore, the results from this study will possibly lead to effective control strategies for not only TbYDV, but also for phytoplasma diseases vectored by O. orientalis .
75 Acknowledgements
This research was funded by Horticulture Australia Limited and the Tobacco Research and Development Corporation with in-kind support from the Department of Primary Industries (DPI), Victoria, Australia. The assistance of Jack Harrington, Anna Harrington, Giuseppe Sacco, growers from NE Victoria, Dr Fiona Constable, Dr Bonny Rowles-Van Rijswijk, Linda Semeraro, Dr Murray Fletcher and colleagues from DPI Rutherglen is gratefully acknowledged
References
Bowyer JW & Atherton JG 1971. Summer death of French bean: new hosts of the pathogen, vector relationship and evidence against mycoplasmal etiology. Phytopathology , 61 :1451-1455.
Creamer R & He X. 1997. Transmission of Sorghum stunt mosaic rhabdovirus by the leafhopper vector, Graminella sonora (Homoptera: Cicadellidae). Plant Disease , 81 :63-65.
Cummins J & Moerkerk M. 1997. Weeds: The Ute Guide : Primary Industries South Australia.
Day MF & Fletcher MJ. 1994. An annotated catalogue of the Australian Cicadelloidea (Hemiptera: Auchenorrhyncha). Invertebrate Taxonomy , 8:1117- 1288.
Day MF, Irzykiewicz H & McKinnon A. 1952. Observations on the feeding of the virus vector Orosius argentatus (Evans), and comparisons with certain other jassids. Australian Journal of Scientific Research , 5:128-142.
76 Evans JW. 1966. The leafhoppers and froghoppers of Australia and New Zealand (Homoptera: Cicadelloidea and Cercopoidea). The Australian Museum Memoir , XII : 1-348.
Fletcher MJ. 2009 and updates Identification keys and checklists for the leafhoppers, planthoppers and their relatives occurring in Australia and neighbouring areas (Hemiptera: Auchenorrhyncha). In http://www.agric.nsw.gov.au/Hort/ascu/start.htm
Fletcher MJ & Larivière M-C. 2009. Anzygina , a new genus for some Australasian microleafhopper species formerly placed in the genus Zygina Fleber (Cicadellidae: Typhlocybinae: Erythroneurini). Australian Journal of Entomology , 48 :164–176.
Ghauri MSK. 1966. Revision of the genus Orosius Distant (Homoptera: Cicadelloidea). Bulletin of the British Museum (Natural History), Entomology , 18 :231-252.
Goodwin PH, Xue BG, Kuske CR & Sears MK. 1994. Amplification of plasmid DNA to detect plant pathogenic mycoplasma like organisms. Annals of Applied Biology , 124 :27-36.
Green MJ, Thompson DA & MacKenzie DJ. 1999. Easy and efficient DNA extraction from woody plants for the detection of phytoplasmas by polymerase chain reaction. Plant Disease Reporter , 83 :482-485.
Grylls NE. 1979. Leafhopper vectors in Australia. In Leafhopper Vectors and Plant Disease Agents (Ed. K. Maramorosch and K.F. Harris.). Academic Press New York:179-214.
Harding RM & Teakle DS. 1985. Mycoplasma-like organisms as causal agents of potato purple top wilt in Queensland. Australian Journal of Agricultural Research , 36 :443-449.
77 Helson GAH. 1942. The leafhopper Thamnotettix argentata Evans, a vector of tobacco yellow dwarf. Journal of Council of Scientific and Industrial Research , 15 :175-184.
Helson GAH. 1950. Yellow dwarf of tobacco in Australia V. Transmission by Orosius argentatus (Evans) to some alternative host-plant. Australian Journal of Agricultural Research , 1:144-147.
Helson GAH. 1951. The transmission of witches broom virus disease of lucerne by the common brown leafhopper, Orosius argentatus (Evans). Australian Journal of Scientific Research, Series B - Biological Science , 4:115-124.
Hill AV. 1937. Yellow dwarf of tobacco in Australia, I. Symptoms. Journal of Council of Scientific and Industrial Research , 10 :228-230.
Hill AV. 1941. Yellow dwarf of tobacco in Australia, II. Transmission by the jassid Thamnotettix argentata (Evans). Journal of the Council for Scientific and Industrial Research , 14 :181-186.
Hill AV. 1950. Yellow dwarf of tobacco in Australia, IV. Some host-plants of the virus. Australian Journal of Agricultural Research , 1:141-143.
Hill AV & Allan MA. 1942. Yellow dwarf of tobacco in Australia, III. Occurrence and agronomic practices. Journal of the Council for Scientific and Industrial Research , 15 :1-13.
Hill AV & Mandryk M. 1954. A study of virus diseases "big bud" of tomato and "yellow dwarf" of tobacco. Australian Journal of Agricultural Research , 5:617- 625.
Hitoshi I & Jutaro H. 1980. Effects of temperature on the transmission of rice waika virus by Nephotettix cinciteps Uhler (Hemiptera: Cicadellidae). Applied Entomology and Zoology , 15 :433-438.
78 Hutton EM & Grylls NE. 1956. Legume 'little leaf', a virus disease of subtropical pasture species. Australian Journal of Agricultural Research , 7:85-97.
Knight WJ. 1975. Deltocephalinae of New Zealand (Homoptera: Cicadellidae). New Zealand Journal of Zoology , 2:169-208.
Knight WJ. 1987. Leafhoppers of the grass-feeding genus Balclutha (Homoptera, Cicadellidae) in the Pacific region. Journal of Natural History , 21 :1173-1223.
Lamp C & Collet F. 1989. Field Guide to Weeds in Australia , Melbourne: Inkata Press.
Moran JR & Rodoni B. 1999. Strategies for the control of tobacco yellow dwarf virus. Report on a consultancy conducted on behalf of the Tobacco Corporation of Victoria.1-5.
Morris BAM, Richardson KA, Haley A, Zhan X & Thomas JE. 1992. The nucleotide sequence of the infectious cloned DNA component of tobacco yellow dwarf virus reveals features of geminiviruses infecting monocotyledonous plants. Virology , 187 :633-642.
Nahid N, Amin I, Mansoor S, Rybicki EP, van der Walt E & Briddon RW. 2008. Two dicot-infecting mastreviruses (family Geminiviridae) occur in Pakistan. Archives of Virology , 153 :1441-1451.
Narhardiyati M & Bailey WJ. 2005. Biology and natural enemies of the leafhopper Balclutha incisa (Matsumura) (Hemiptera: Cicadellidae: Deltocephalinae) in south-western Australia. Australian Journal of Entomology , 44:104-109.
Osmelak JA. 1986. Assessment of various insecticides for the control of the vector Orosius argentatus (Evans) (Homoptera: Cicadellidae) and tomato big bud diseases. Department of Agricultural and Rural Affairs. Research Report : 1-32.
79 Paddick RG & French FL. 1972. Suppression of tobacco yellow dwarf with systematic organophosphorus insecticide. Australian Journal of Experimental Agriculture and Animal Husbandry , 12 :331-334.
Paddick RG, French FL & Turner PL. 1971. Control of leafhopper-borne plant disease possibly due to direct action of systemic biocides on mycoplasmas. Plant Disease Reporter , 55 :291-293.
Parsons WT & Cuthbertson EG. 1992. Noxious Weeds of Australia , Melbourne, Sydney: Inkata Press.
Pilkington LJ, Gurr GM, Fletcher MJ, Nikandrow A & Elliott E. 2004. Vector status of three leafhopper species for Australian lucerne yellows phytoplasma. Australian Journal of Entomology , 42 :366-373.
Strindberg S & Buckland ST. 2004. Zigzag survey designs in line transect sampling. Journal of Agricultural, Biological and Environmental Statistics 9:443- 461.
Thomas JE & Bowyer JW. 1979. The purification and identification of tobacco yellow dwarf and Bean summer death viruses. Australasian Plant Pathology , 8:36-38.
Thomas JE & Bowyer JW. 1980. Properties of tobacco yellow dwarf and bean summer death viruses. Phytopathology , 70 :214-217.
Tr ębicki P, Powell KS & Baxter G. 2007. Epidemiology and management of leafhopper that transmit a unique virus In: Sustainable tobacco production. Ed. by Baxter G, Hayes G, Harrington A, Harrington J, Sacco G. Department of Primary Industries, Final Report, TBO4001, 132. van Rijswijk B, Rodoni BC, Baxter G & Moran JR. 2002. Managing tobacco yellow dwarf virus. Phase 1: Developing a diagnostic tool. Phase 2: Beginning to
80 understand the epidemiology of TYDV . Department of Natural Resources and Environment , pp. 48. van Rijswijk B, Rodoni BC, Revill PA, Thomas JE, Moran JR & Harding RM. 2004. Analysis of variability in partial sequences of genomes of Tobacco yellow dwarf virus isolates. Australasian Plant Pathology , 33 :367-370.
Witt ABR & Edwards PB. 2000. Biology, distribution, and host range of Zygina sp. (Hemiptera: Cicadellidae), a potential biological control agent for Asparagus asparagoides . Biological Control , 18 :101-109.
81
82 CHAPTER 4
DIVERSITY OF CICADELLIDAE IN AGRICULTURAL PRODUCTION AREAS IN THE OVENS VALLEY, NORTHEAST VICTORIA, AUSTRALIA
Piotr Tr ębicki 1, 2 , Rob M. Harding 1, Brendan Rodoni 3, Gary Baxter 4
& Kevin S. Powell 2
1Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Queensland, GPO Box 2434, Brisbane, 4001, Australia. 2Department of Primary Industries, Biosciences Research Division, RMB 1145, Chiltern Valley Road, Rutherglen, Victoria, 3685, Australia. 3Department of Primary Industries, Private Bag 15, Ferntree Gully Delivery Centre, Knoxfield, Victoria, 3180, Australia. 4TAFCO Rural Supplies, Great Alpine Highway, Myrtleford, Victoria, 3737, Australia.
Journal of Australian Entomology (2010), 49, 213-220
83 STATEMENT OF AUTHORSHIP
Piotr Tr ębicki (principal author): Executed the work (collected samples, designed and conducted all laboratory experiments, analysed and interpreted data) and wrote initial manuscript.
Signed ……………………………………. Date …18-12-09…………………..
Rob Harding: Conceived project idea, critically interpreted data and contributed to final manuscript.
Signed ……………………………………. Date …18-12-09………….………..
Brendan Rodoni: Conceived project idea, critically interpreted data and contributed to final manuscript.
Signed ……………………………………. Date …18-12-09…………………..
Gary Baxter: Conceived project idea, critically interpreted data and contributed to final manuscript.
Signed ……………………………………. Date …18-12-09…………………..
Kevin Powell: Conceived project idea, supervised the work, critically interpreted data and contributed to final manuscript.
Signed ……………………………………. Date …18-12-09……..……………..
84
Abstract
There is a paucity of data on the distribution of Cicadellidae (leafhoppers) in Australia. This study quantifies the relative abundance, seasonal activity and diversity of leafhoppers in the Ovens Valley region of Northeast Victoria, Australia. Species diversity and abundance was assessed at four field sites in and around the field borders of commercially grown tobacco crops using three sampling techniques (pan trap, sticky trap and sweep net). Over 51000 leafhopper samples were collected, with 57 species from 11 subfamilies and 19 tribes identified. Greater numbers and diversity of leafhoppers were collected in yellow pan traps . The predominant leafhopper collected was Orosius orientalis (Matsumura). Twenty-three leafhopper species were recorded for the first time in Victoria and eight economically important pest species were recorded. Seasonal activity of selected leafhopper species, covering two sampling seasons, is presented.
85 Introduction
Leafhoppers (Hemiptera: Cicadellidae) are herbivorous insects and many species are economically important pests of agricultural crops in Australia and worldwide (Day & Fletcher 1994). These sap-sucking pest species cause serious damage either directly through feeding or indirectly by transmitting plant pathogens including viruses and phytoplasmas (Backus et al. 2005; Gray & Banerjee 1999; Grylls 1979; Weintraub & Beanland 2006). One of the most important and relatively well-studied leafhopper vectors in Australia is Orosius orientalis (Matsumura) which has a broad host range (Tr ębicki et al . 2010). It is a key vector of many viruses and phytoplasmas in Australia and worldwide. In Australia, O. orientalis is responsible for transmitting Tobacco yellow dwarf virus (TbYDV, genus Mastrevirus, family Geminiviridae) to beans ( Phaseolus vulgaris L.) and tobacco ( Nicotiana tabacum L.) (Hill 1941; 1950; Thomas & Bowyer 1980; van Rijswijk et al. 2002), resulting in economically important losses due to diseases (Tr ębicki et al. 2010a). It also transmits numerous phytoplasmas which cause a range of serious diseases in legumes (Hutton & Grylls 1956), tomato (Hill 1941), lucerne (Helson 1951; Pilkington et al. 2004a) and potato (Grylls 1979; Harding & Teakle 1985) and is also associated with the spread of diseases in papaya, and grapes (Beanland, 2002; Padovan & Gibb, 2001). Other economically important leafhopper pests previously recorded in Australia include Austroasca viridigrisea (Paoli) which causes leaf distortion and stunting in vegetables (Page 1983; Mensah 1996), Austroagallia torrida Evans which is a vector of Rugose leaf curl virus (Grylls 1954; Grylls et al. 1947) and Batracomorphus angustatus (Osborn) a vector of the phytoplasmas which cause tomato big bud and potato purple top wilt disease (Grylls 1979). Although insecticides have been used to control many pest leafhoppers, their use does not necessarily provide effective disease control (Paddick & French 1972; Paddick et al. 1971) since this requires an understanding of the insect population dynamics to ensure the optimal timing of insecticide application (Broadbent 1957; Perring et al. 1999). In many instances, however, the most
86 suitable sampling method to monitor insect population abundance and migration has not been determined. This paper describes a study of a diverse leafhopper community in and around tobacco production areas in the Ovens Valley region of Northeast Victoria over two cropping seasons. The main aims of the study were to qualitatively analyse leafhopper diversity, identify the most prevalent leafhopper species and compare the efficiency of three sampling methods to monitor the seasonal activity of both native and pest leafhopper species.
Materials and methods
Study sites This field-based study was conducted during two tobacco (Nicotiana tabacum L.) growing seasons, 2005-06 and 2006-07, at four sites (A, B, C and D) on commercial farms located near Myrtleford in Northeast Victoria, Australia. Tobacco has been the predominant cash crop grown at all study sites for the most part of the twentieth century. Sites A and B were neighbouring farms located in the lower Ovens Valley near Whorouly (36°29'23''S, 146°35'48''E 210 masl), while Site C was located between Ovens and Eurobin (36°37'12''S, 146°48'31''E, 246 masl). Site D was located 6 km from Porepunkah (36°39'46''S, 146°14'56''E 320 masl).
Sampling methods From December 2005 to June 2006 (season 1) and September 2006 to June 2007 (season 2), the relative seasonal abundance and activity of leafhoppers was monitored at all four sites using three different sampling methods; yellow sticky trap, yellow pan trap and sweep net. Sticky traps and pan traps were used to monitor weekly leafhopper activity and sweep nets were used to monitor leafhopper abundance. One sticky trap coated with Tanglefoot and one pan trap was placed every 10 m along a 100 m transect. Pan traps were placed between sticky traps 5 m apart. Sticky traps (Bugs for Bugs, Australia; 32 x 10 cm) were attached to wooden stakes 20 cm above the plant canopy. Ten sticky and ten pan
87 traps were used per site. At weekly intervals, all leafhoppers were removed and transported to the laboratory for counting and identification. Each trap was processed separately and on the same day of collection. Pan traps (Moericke 1951; 1955) were made from round, shallow plastic containers (10 x 34 cm diameter) with a pre-moulded groove on the side to drain the liquid. All pan traps were painted using yellow Dulux® spray paint. Yellow pan traps were placed on the ground and filled to 90% capacity with 5-7% saline fixative solution and a few drops of commercial dishwashing detergent to act as a surfactant. The liquid level in all traps was checked between sample collections and topped up as required. Traps were emptied weekly, by straining all contents through a very fine sieve, and transferred into 70% ethanol prior to segregation and identification. Each trap was processed separately and the containers with specimens were transferred to a Petri dish with 70% ethanol and counted. Due to the large numbers of leafhoppers from each trap, all counting was done using a digital cell Grale counter. Weekly sweep net sampling was conducted. The samples were collected in the same plot area adjacent to tobacco field using the sampling procedure described by Tr ębicki et al . (2010a; 2010b). Sweep nets (38 cm diameter, very fine mesh) were used by swinging the net through the plant canopy, represented by a range of hosts (see Tr ębicki et al . 2010a); at 180 ° along a 100 m distance and each sweep was approximately one metre. Any potential influence of sweep net catches on sticky trap and pan trap was minimised as described in Tr ębicki et al. (2010b). One hundred sweeps per site per sampling date were done and trapped insects were placed into a killing jar containing ethyl acetate for 10-15 minutes then transferred into zip-lock plastic bags. To size differentiate samples, five different laboratory test sieves were used. Each sieve was emptied into a white plastic tray and, using a magnifying glass and forceps, all leafhoppers were transferred to a Petri dish for identification and counting using a stereo microscope.
Identification All adult leafhoppers were identified based on morphological characteristics using identification keys (Evans 1966; Fletcher 2009; Ghauri 1966). For most leafhopper species, a detailed examination of the internal structure of male
88 genitalia was also necessary due to difficulties in identification using external characteristics. For this purpose, the abdominal apex was carefully removed with entomological pins, placed in heated 10% potassium hydroxide to macerate the muscle and soft connective tissue was removed making the internal structures clearly visible (Oman 1949). Reference material of selected described species are housed at DPI Rutherglen and undescribed species at the Orange Agricultural Institute, DPI NSW.
Meteorological data Meteorological data for both collecting seasons were obtained from the Bureau of Meteorology, Wangaratta, Victoria and the SILO database (http://www.bom.gov.au/silo/ ).
Results
Leafhopper diversity and abundance Leafhopper monitoring was conducted over two collecting seasons; from December 2005 to June 2006 and from September 2006 to June 2007. During this period, a combined total of 51,427 leafhoppers was recorded from the four sites using three sampling methods. Fifty seven described species represented by 11 subfamilies and 19 tribes (Table 4.1) were identified. Deltocephalinae and Typhlocybinae were the most abundant subfamilies with 31 and 10 species recorded, respectively. In contrast, the Agalliinae, Iassinae, Euacanthellinae and Xestocephalinae subfamilies were each represented by only a single species (Table 4.1). The most abundant species collected was Orosius orientalis (Matsumura) which represented 52% (n=26,954) of the total sample size. Other species collected in relatively high numbers were Anzygina zealandica (Myers) (number of leafhopper specimens n=6,505), Maiestas knighti (Webb & Viraktamath) (n=6,358), Xestocephalus tasmaniensis (Evans) (n=4,482), Nesoclutha phryne
89 (Kirkaldy) (n=3,384), Arawa pulchra (Knight) (n=791), Batracomorphus angustatus (Osborn) (n=763), Austroasca viridigrisea (Paoli) (n=366) and Balclutha frontalis (Ferrari) (n=165) (Figure 4.1). In addition, two economically important pest species, Austroagallia torrida (Evans) and Ribautiana ulmi (Linnaeus) were recorded, albeit in relatively low abundance. The remaining non- pest species were recorded in relatively low numbers (< 60) with 20 species recorded on only one or two occasions and generally only from single sites (Table 4.1). Twenty-three species collected had not been recorded previously in Victoria (Table 4.1), while 15 other, as yet undescribed, species (M. Fletcher pers. comm.) were also collected. Pan traps recorded the largest abundance of Cicadellidae at all field sites. Over both seasons, 78% of Cicadellidae were collected using pan traps with 13% and 9% recorded using sticky traps and sweep nets, respectively. The greatest species diversity (n=43) was also recorded using pan traps (Figure 4.2), whereas similar species numbers were collected using sticky traps (n=32) and sweep nets (n=33). Nineteen species were common to all monitoring methods (Figure 4.2) while two species, Horouta spp and Scaphoideus spp, were only recorded using pan traps. There was some general variation in leafhopper abundance and diversity between sites with sites B and C recording the highest total number of leafhoppers as well as higher species diversity (data not shown). This site–to–site variation may have been influenced by climatic and vegetation differences. Sites A and B had relatively higher average temperatures and lower rainfall than sites C and D. The diversity and abundance of vegetation was also marginally different between sites (Tr ębicki et al . 2010a).
90
Figure 4.1 Diversity and abundance of leafhopper species collected from four sites, using sweep net, pan and sticky traps, in the Ovens Valley, Northeast Victoria, over two seasons during 2005-2007.
91 Table 4.1 Diversity and abundance of leafhopper species recorded using three sampling methods (pan trap, sticky trap and sweep net) at four field sites in the Ovens Valley, Northeast Victoria during two collection seasons during 2005-2007.
Subfamily: Tribe Species Sampling method Sweep Pan Sticky
net trap trap Agalliinae Austroagallia torrida (Evans) 15 21 19 Deltocephalinae: Athysanini Arawa detracta (Walker)* 16 Arawa novella Metcalf* 15 29 Arawa pulchra Knight 56 727 8 Exitianus nanus Distant 2 Exitianus plebeius (Kirkaldy) 14 311 20 Limotettix incertus (Evans) 3 11 10 Thamnophryne nysias Kirkaldy* 11 16 6 Deltocephalinae: Chiasmini Chiasmus varicolor (Kirkaldy)* 18 31 Deltocephalinae: Deltocephalini Deltocephalus viridellus Evans* 4 Horouta aristarche (Kirkaldy)* 3 Horouta austrina (Kirkaldy)* 11
92 Horouta lotis (Kirkaldy) 22 Horouta perparvus (Kirkaldy) 594 Horouta spinosa Fletcher* 7 Maiestas knighti Webb & Viraktamath 16 6062 280 Maiestas vetus (Knight) 2 Deltocephalinae: Macrostelini Balclutha chloe (Kirkaldy)* 7 2 1 Balclutha incisa (Matsumura) 12 9 Balclutha lucida (Butler)* 3 1 2 Balclutha frontalis (Ferrari) 145 9 11 Balclutha saltuella (Kirschbaum)* 9 6 Nesoclutha phryne (Kirkaldy) 434 2457 493 Deltocephalinae: Opsiini Opsius stactogalus Fieber 2 Orosius canberrensis (Evans) 18 39 Orosius orientalis (Matsumura) 2842 20465 3638 Deltocephalinae: Paralimnini Diemoides smithtoniensis Evans 2 4 9 Euleimonios kirkaldyi Fletcher 5 Soractellus nigrominutus Evans* 5 Deltocephalinae: Scaphoideini Scaphoideus foshoi Fletcher & Semeraro* 18 Scaphoideus obscurus Fletcher & Semeraro* 3
93 Scaphoideus pristidens Kirkaldy* 6 Euacanthellinae: Euacanthellini Euacanthella palustris (Evans)* 7 Eurymelinae: Eurymelini Eurymeloides musgravei (Evans) 16 Eurymeloides pulchra (Signoret) 7 8 15 Iassinae: Iassini Batracomorphus angustatus (Osborn) 38 290 435 Tartessinae: Stenocotini Smicrocotis obscura Kirkaldy 2 Stenocotis depressa (Walker) 2 Tartessinae: Tartessini Alotartessus iambe (Kirkaldy)* 3 Tenuitartessus blundellensis (Evans)* 5 Tartessinae: Thymbrini Putoniessa dorsalis (Walker) 2 17 Putoniessa nigra (Walker) 8 5 Putoniessa nigrella Evans 3 22 Putoniessa rieki Stevens * 4 Typhlocybinae: Dikraneurini Aneono australensis (Kirkaldy)* 7 8 13 Kahaono pallida Evans 4 16 Kahaono viridis Evans 7 22 Kahaono wallacei Evans* 23 Typhlocybinae: Empoascini Austroasca viridigrisea (Paoli) 86 106 174 Kybos lindbergi (Linnavuori)* 1 2
94 Typhlocybinae: Erythroneurini Zygina evansi (Ross) 9 8 23 Anzygina sidnica (Kirkaldy) 17 2 Anzygina zealandica (Myers) 652 4771 1082 Typhlocybinae: Typhlocybini Ribautiana ulmi (Linnaeus) 2 Ulopinae: Cephalelini Alocephalus ianthe (Kirkaldy) 2 3 Ulopinae: Ulopini Kahavalu gemma Kirkaldy* 2 Xestocephalinae Xestocephalus tasmaniensis Evans 12 4343 127
*indicates species not previously recorded in Victoria. Species in bold font represent economically important pest species.
95
Figure 4.2 Total number of leafhopper species collected over two growing seasons at four sites in the Ovens Valley region, Northeast Victoria, using three sampling methods (pan trap, sticky trap and sweep net). Overlapping areas indicate the number of leafhopper species common for each trap type.
96
Seasonal leafhopper activity When insect population data were analysed over two collection seasons, the greatest numbers of native and economically important species (Figure 4.3) were recorded in spring and summer. Generally, trimodal peaks of activity were observed for A. zealandica, O. orientalis and M. knighti . The initial peak predominantly occurred early in the season during spring (late September), the second late November to December while the third peak occurred in either January to February or March to April depending on the species. For B. angustatus and X. tasmaniensis , only two peaks of population activity were recorded in November and late January, and December and late April, respectively (Figure 4.3). Apart from B. angustatus and X. tasmaniensis , most leafhopper species had higher summer peak activity during the first season compared to the second season. Some leafhopper species, however, did not occur in sufficient numbers to show noticeable trends in seasonal activity (data not shown). Rainfall and temperature data were analysed over the two seasons (2005- 2007) to assess the effect of climatic conditions on insect populations. These analyses revealed that average rainfall from all sites was higher during the first collection season (490 mm) compared to the second season (319 mm), while the average maximum temperatures for the first season (24.6°C) were lower than the second season (26.6°C).
97
Figure 4.3 Seasonal activity and abundance of six selected leafhopper species collected over two seasons, during 2005-2007 in the Ovens Valley region of Northeast Victoria. Pooled data is shown from four field sites for weekly sample dates.
98
Discussion
A range of Cicadellidae is known to be present in Victoria, many of which are economically important pest species and vectors of pathogens of economically important crops. This study is the first to quantify the relative abundance and diversity of leafhoppers in the Ovens Valley, Northeast Victoria. The research was conducted on commercial tobacco farms and provided a qualitative and quantitative understanding of the Cicadellidae community in the region. Three sampling methods were utilised over two consecutive growing seasons to maximise insect collection data. Orosius orientalis was found to be the most abundant species of leafhoppers collected and is known to be a major economic pest in the region (Tr ębicki et al . 2010b). Several other leafhoppers were also found to occur in relatively high numbers, including Anzygina zealandica , Nesoclutha phryne , Maiestas knighti and Batracomorphus angustatus . Anzygina zealandica is primarily a grass-feeding leafhopper but has also been recorded on shrubs and trees (Knight 1976; Moir et al. 2003) while N. phryne (Kirkaldy) = N. pallida (Evans) is a vector of Chloris striate mosaic virus , Paspalum striate mosaic virus and Cereal chlorotic mottle rhabdovirus (Greber 1977a; Greber 1977b; Grylls 1963). Maiestas knighti and B. angustatus are both common in many crops and are important vectors of the phytoplasmas causing tomato big bud and potato purple top wilt (Grylls 1979), respectively. Austroasca viridigrisea , a common pest of potatoes, tomatoes, beans, tobacco and lucerne (Page 1979; 1983), was also frequently recorded. Further taxonomic work is needed for an additional 15 unidentified leafhopper species that were collected in this study, and which are likely to be described species. For example, currently in Australia in the Erythroneurini tribe, there are several undescribed species assigned to Anzygina spp., Empoascanara spp. and Pettya spp. (Fletcher 2009 and updates). The fact that 41% of all species identified in this study had not previously been recorded in Victoria highlights the limitations of current leafhopper distribution data in Australia. There has been limited research directed towards investigating the population dynamics and diversity of leafhoppers in Australia
99
over the past few decades. Furthermore, this has been restricted to selected crops including lucerne (Helson 1951; Pilkington et al. 2004b), tomato (Osmelak 1986; Osmelak & Fletcher 1988) and tobacco (Helson 1942; 1950; Hill 1941; Hill & Helson 1949; Tr ębicki et al. 2010a) with most studies targeting a single leafhopper species, particularly O. orientalis. This is the first systematic study conducted over successive seasons on a diverse range of Cicadellidae collected from vegetation around the borders of tobacco fields in Northeast Victoria. Of the three sampling methods evaluated in this study, both the largest number and greatest diversity of leafhopper species was recorded using the pan trap. Despite obvious differences in the numbers of the most common leafhoppers collected, the highest numbers were recorded around the same time in all traps, especially for O. orientalis . In a recent study, Tr ębicki et al. (2010b) reported that pan traps and sticky traps were the most suitable methods for the monitoring of O. orientalis , while sweep netting is the preferable monitoring option where fresh samples are needed for pathogen testing. Two additional trapping methods, incandescent light and suction trap, have also been proven effective in collecting leafhoppers outside cropping areas (Osmelak 1987). Although these two methods and collecting using an insect aspirator were also used in the first few weeks of the present study, both were subsequently discontinued due to relatively low catch numbers (data not shown). In previous studies on Hemiptera from understorey or canopy plants, chemical knockdown, vacuum sampling and beating have proven more suitable than sticky trap, hand collecting and branch clipping (Moir et al . 2005). In our study, clear differences were found in both the diversity and abundance of leafhopper species detected using sticky and pan traps. Trap positioning, in relation to surrounding vegetation, is likely to be a critical factor in determining the species collected. Pan traps were placed on the ground, whereas sticky traps were raised above the vegetation. As such, larger numbers of leafhopper species displaying short distance jumps and low level flight activity, such as N. phryne, O. orientalis and A. zealandica species (Hosking & Danthanarayana 1988), might be expected to be trapped in pan traps. This notion is supported by a study which showed that increases in the above-ground height of yellow sticky traps resulted in a decrease in the numbers of trapped O. orientalis and A. torrida (Pilkington et al. 2004a).
100
Although the widest diversity of leafhoppers was obtained using pan traps, this method was also relatively time and labour intensive with respect to collecting and processing samples. For example, the high temperature and evaporation rates occurring during the summer months not only necessitated the regular top-up of the trap collection fluid but also resulted in the rapid decomposition of some specimens which hindered their identification. The processing of sticky traps was also relatively time consuming and identification of specimens caught in Tanglefoot was somewhat difficult. Despite the sweep net proving the least efficient method for collecting most leafhopper species, especially O. orientalis, it was the most convenient in terms of ease of collection and sample processing. Clearly, the use of several trap types is ideally needed to accurately monitor population abundance, seasonal activity and diversity of a number of different leafhopper species. Due to time and economic constraints, however, this option is not always practical, especially for farmers implementing IPM who often target specific insect pests in monoculture environments. Regardless of the trapping method(s) used, an understanding of the abiotic and biotic factors influencing leafhopper species abundance in a particular region will enable the development of targeted insect management strategies and subsequent strategies for effective disease control. Overall this study highlighted that a diverse range of leafhoppers, are present in the Ovens Valley region and some of predominant species are economically important vectors of phytoplasmas and viruses and pan traps were particularly useful in monitoring seasonal activity.
Acknowledgements
We wish to acknowledge the technical assistance of Jack and Anna Harrington, and Giuseppe Sacco from Department of Primary Industries (DPI) Ovens, Dr Murray Fletcher (DPI Orange, NSW) and Linda Semeraro (DPI Knoxfield, Victoria) for their assistance with taxonomic confirmation. This research was
101
funded by Horticulture Australia Limited and the Tobacco Research and Development Corporation with in-kind support from DPI, Victoria, Australia.
References
Backus EA, Serrano MS & Ranger CM. 2005. Mechanisms of hopperburn: an overview of insect taxonomy, behaviour and physiology. Annual Review of Entomology 50 , 125-151.
Beanland L. 2002. Developing Australian Grapevine Yellows Management Strategies. GWRDC Final Report DNR00/02 Australia: 1-24.
Broadbent L. 1957. Insecticidal control of the spread of plant viruses. Annual Review of Entomology 2, 339-354.
Day MF & Fletcher MJ. 1994. An annotated catalogue of the Australian Cicadelloidea (Hemiptera: Auchenorrhyncha). Invertebrate Taxonomy 8, 1117- 1288.
Evans JW. 1966. The leafhoppers and froghoppers of Australia and New Zealand (Homoptera: Cicadelloidea and Cercopoidea). The Australian Museum Memoir XII , 1-348.
Fletcher MJ. 2009. Identification keys and checklists for the leafhoppers, planthoppers and their relatives occurring in Australia and neighbouring areas (Hemiptera: Auchenorrhyncha). Agricultural Scientific Collections Trust, Orange Agricultural Institute, NSW DPI, Orange, New South Wales. (ed MJ Fletcher) In: http://www.agric.nsw.gov.au/Hort/ascu/start.htm.
Ghauri MSK. 1966. Revision of the genus Orosius Distant (Homoptera: Cicadelloidea). Bulletin of the British Museum (Natural History), Entomology 18 , 231-252.
102
Gray SM & Banerjee N. 1999. Mechanisms of arthropod transmission of plant and animal viruses. Microbiology and Molecular Biology Reviews . American Society of Microbiology 63 , 128-148.
Greber RS. 1977a. Cereal chlorotic mottle virus (CCMV), a rhabdovirus of Gramineae transmitted by the leafhopper Nesoclutha pallida . Australasian Plant Pathology Society Newsletter 6, 17.
Greber RS. 1977b. Transmission of Chloris striate mosaic disease from field- infected cereals. Australasian Plant Pathology Society Newsletter 6, 4.
Grylls NE. 1954. Rugose leaf curl - a new virus disease transovarially transmitted by the leafhopper Austroagallia torrida . Australian Journal of Biological Sciences 7, 47-58.
Grylls NE. 1963. A striate mosaic virus disease of grasses and cereals in Australia, transmitted by the cicadellid Nesoclutha obscura . Australian Journal of Agricultural Research 14 , 143-153.
Grylls NE. 1979. Leafhopper vectors and the plant disease agents they transmit in Australia. In: Leafhopper Vectors and Plant Disease Agents (eds K Maramorosch & KF Harris) 179–214. Academic Press, New York.
Grylls NE, Waterford CJ, Filshie BK & Beaton CD. 1947. Electron microscopy of Rugose leaf curl virus in red clover, Trifolium pratense and in the leafhopper vector Austroagallia torrida . Journal of General Virology 23 , 179-183.
Harding RM & Teakle DS. 1985. Mycoplasma-like organisms as causal agents of potato purple top wilt in Queensland. Australian Journal of Agricultural Research 36 , 443-449.
Helson GAH. 1942. The leafhopper Thamnotettix argentata Evans, a vector of tobacco yellow dwarf. Journal of Council of Scientific and Industrial Research 15 , 175-184.
103
Helson GAH. 1950. Yellow dwarf of tobacco in Australia V. Transmission by Orosius argentatus (Evans) to some alternative host-plant. Australian Journal of Agricultural Research 1, 144-147.
Helson GAH. 1951. The transmission of witches broom virus disease of lucerne by the common brown leafhopper, Orosius argentatus (Evans). Australian Journal of Scientific Research, Series B - Biological Science 4, 115-124.
Hill AV. 1941. Yellow dwarf of tobacco in Australia, II. Transmission by the jassid Thamnotettix argentata (Evans). Journal of the Council for Scientific and Industrial Research 14 , 181-186.
Hill AV. 1950. Yellow dwarf of tobacco in Australia, IV. Some host-plants of the virus. Australian Journal of Agricultural Research 1, 141-143.
Hill AV & Helson GA. 1949. Distribution in Australia of three virus diseases and of their common vector Orosius argentatus (Evans). Journal of the Australian Institute of Agricultural Science. 15 , 160-161.
Hosking JR & Danthanarayana W. 1988. Low level flight activity of Nesoclutha pallida (Evans), Orosius argentatus (Evans) and Zygina zealandica (Myers) (Hemiptera: Cicadellidae) in Southern Victoria. Journal of the Australian Entomological Society 27 , 241-249.
Hutton EM & Grylls NE. 1956. Legume 'little leaf', a virus disease of subtropical pasture species. Australian Journal of Agricultural Research 7, 85-97.
Knight WJ. 1976. Typhlocybinae of New Zealand (Homoptera: Cicadellidae). New Zealand Journal of Zoology 3, 71-87.
Moericke V. 1951. Eine Farbfalle zur Kontrolle des Fluges von Blattlausen, insbesondere der Pfirsichblattlaus Myzodes persicae (Sulz). Nachrichtenblatt des Deutschen Pflanzenschutzdienste. Stuttgart 3. 23-24
Moericke V. 1955. Über die Lebensgewohnheiten der geflügelten Blattläuse (Aphidina) unter besonderer Berücksichtigung des Verhaltens beim Landen. Zeitschrift für Angewandte Entomologie 37 , 29-91.
104
Moir ML, Majer JD & Fletcher MJ. 2003. New records for Hemiptera species in Western Australia. Records of the Western Australian Museum 21 , 353-357.
Moir ML, Brennan KEC, Majer JD, Fletcher MJ & Koch JM. 2005. Toward an optimal sampling protocol for Hemiptera on understorey plants. Journal of Insect Conservation 9, 3–20
Oman PW. 1949. The Nearctic leafhoppers (Homoptera: Cicadellidae): a generic classification and check list. Memoirs of the Entomological Society of Washington 3, 1-253.
Osmelak JA. 1986. Assessment of various insecticides for the control of the vector Orosius argentatus (Evans) (Homoptera: Cicadellidae) and tomato big bud diseases. Department of Agricultural and Rural Affairs, Research Report 1-32.
Osmelak JA. 1987. A comparison of five different traps for monitoring leafhopper activity (Homoptera). General and Applied Entomology 19 , 52-56.
Osmelak JA & Fletcher MJ. 1988. Auchenorrhyncha (Hemiptera: Cicadellidae, Fulgoroidea, Cercopidae) trapped near tomato crops at Tatura, Victoria. General and Applied Entomology 20 , 52-56.
Paddick RG & French FL. 1972. Suppression of tobacco yellow dwarf with systematic organophosphorus insecticide. Australian Journal of Experimental Agriculture and Animal Husbandry 12 , 331-334.
Paddick RG, French FL & Turner PL. 1971. Control of leafhopper-borne plant disease possibly due to direct action of systemic biocides on mycoplasmas. Plant Disease Reporter 55 , 291-293.
Padovan AC & Gibb KS. 2001 Epidemiology of phytoplasma diseases in papaya in Northern Australia. Journal of Phytopathology 149 , 649-658.
Page FD. 1979. The immature stages of Austroasca viridigrisea (Paoli) (Homoptera: Cicadellidae: Typhlocybinae). Australian Journal of Entomology 18 , 111-114.
105
Page FD. 1983 Biology of Austroasca viridigrisea (Paoli) (Hemiptera: Cicadellidae). Australian Journal of Entomology 22 , 149-153.
Perring TM, Gruenhagen NM & Farrar CA. 1999. Management of plant viral diseases through chemical control of insect vectors. Annual Review of Entomology 44 , 457-481.
Pilkington LJ, Gurr GM, Fletcher MJ, Elliott E, Nikandrow A & Nicol HI. 2004a. Reducing the immigration of suspected leafhopper vectors and severity of Australian lucerne yellows disease. Australian Journal of Experimental Agriculture 44 , 983–992
Pilkington LJ, Gurr GM, Fletcher MJ, Nikandrow A & Elliott E. 2004b. Vector status of three leafhopper species for Australian lucerne yellows phytoplasma. Australian Journal of Entomology 42 , 366-373.
Thomas JE & Bowyer JW. 1980. Properties of tobacco yellow dwarf and bean summer death viruses. Phytopathology 70 , 214-217.
Tr ębicki P, Harding RM & Powell KS. 2009. Antimetabolic effects of Galanthus nivalis agglutinin and wheat germ agglutinin on nymphal stages of the common brown leafhopper using a novel artificial diet system. Entomologia Experimentalis et Applicata 131 , 99-105.
Tr ębicki P, Harding RM, Rodoni B, Baxter G & Powell KS. 2010a. Vectors and alternative hosts of TbYDV in south-eastern Australia. Annals of Applied Biology 157 , 13-24.
Tr ębicki P, Harding RM, Rodoni B, Baxter G & Powell KS. 2010b. Seasonal activity and abundance of Orosius orientalis (Hemiptera: Cicadellidae) at agricultural sites in Southeastern Australia. Journal of Applied Entomology 134 , 91-97.
Weintraub PG & Beanland L. 2006. Insect vectors of phytoplasmas. Annual Review of Entomology 51 , 91-111.
106
van Rijswijk B, Rodoni BC, Baxter G & Moran JR. 2002. Managing tobacco yellow dwarf virus. Phase 1: Developing a diagnostic tool. Phase 2: Beginning to understand the epidemiology of TYDV. In: Department of Natural Resources and Environment pp.48. Tobacco Research and Development Corporation Final Report 1999/2001 of Project DAV 35T.
107
108
CHAPTER 5
SEASONAL ACTIVITY AND ABUNDANCE OF OROSIUS ORIENTALIS (HEMIPTERA: CICADELLIDAE) AT AGRICULTURAL SITES IN SOUTHEASTERN AUSTRALIA
Piotr Tr ębicki 1, 2 , Rob M. Harding 1, Brendan Rodoni 3, Gary Baxter 4
& Kevin S. Powell 2
1Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Queensland, GPO Box 2434, Brisbane, 4001, Australia. 2Department of Primary Industries, Biosciences Research Division, RMB 1145, Chiltern Valley Road, Rutherglen, Victoria, 3685, Australia. 3Department of Primary Industries, Private Bag 15, Ferntree Gully Delivery Centre, Knoxfield, Victoria, 3180, Australia. 4TAFCO Rural Supplies, Great Alpine Highway, Myrtleford, Victoria, 3737, Australia.
Journal of Applied Entomology (2010), 134, 91-97.
109
STATEMENT OF AUTHORSHIP
Piotr Tr ębicki (principal author): Executed the work (collected samples, designed and conducted all laboratory experiments, analysed and interpreted data) and wrote initial manuscript.
Signed ……………………………………. Date …18-12-09.…………………..
Rob Harding: Conceived project idea, critically interpreted data and contributed to final manuscript.
Signed ……………………………………. Date …18-12-09..…………………..
Brendan Rodoni: Conceived project idea, critically interpreted data and contributed to final manuscript.
Signed ……………………………………. Date …18-12-09...…………………..
Gary Baxter: Conceived project idea, critically interpreted data and contributed to final manuscript.
Signed ……………………………………. Date …18-12-09...…………………..
Kevin Powell: Conceived project idea, supervised the work, critically interpreted data and contributed to final manuscript.
Signed ……………………………………. Date …18-12-09...…………………..
110
Abstract
Orosius orientalis is a leafhopper vector of several viruses and phytoplasmas affecting a broad range of agricultural crops. Sweep net, yellow pan trap and yellow sticky trap collection techniques were evaluated. Seasonal distribution of O. orientalis was surveyed over two successive growing seasons around the borders of commercially grown tobacco crops. Orosius orientalis seasonal activity as assessed using pan and sticky traps was characterised by a trimodal peak and relative abundance as assessed using sweep nets differed between field sites with peak activity occurring in spring and summer months. Yellow pan traps consistently trapped a higher number of O. orientalis than yellow sticky traps.
111
Introduction
Orosius orientalis (Matsumura), the common brown leafhopper [synonym = O. argentatus (Evans)] belongs to the large and diverse non-endopterygote Hemipteran order and is further classified into the Auchenorrhyncha suborder and Cicadellidae (leafhoppers) family (Day & Fletcher 1994; Dietrich 2005; Fletcher 2009; Oman et al. 1990). Orosius orientalis is an important vector of many viruses and phytoplasmas in Australia and worldwide. In Australia, O. orientalis is responsible for transmitting Tobacco yellow dwarf virus (TbYDV, genus Mastrevirus , family Geminiviridae ) to beans ( Phaseolous vulgaris L.) and tobacco ( Nicotiana tabacum L.) (Hill 1941; Hill 1950; Thomas & Bowyer 1980; van Rijswijk et al. 2002) resulting in economically important production and quality losses of up to 4.5 million AUD annually (Tr ębicki et al. 2007). Orosius orientalis also transmits numerous phytoplasmas which cause a range of important diseases including legume little leaf (Hutton & Grylls 1956), tomato big bud (Hill 1941), lucerne witches broom (Helson 1951), potato purple top wilt (Grylls 1979; Harding & Teakle 1985) and Australian lucerne yellows (Pilkington et al. 2004a). Orosius orientalis is active around the borders of tobacco fields where it predominates on several weed species including Trifolium repens , Cryptostemma calendulaceae , Plantago lanceolata , Malva parviflora , Amaranthus retroflexus and Raphanus raphanistrum (Tr ębicki et al. 2010). Some of these primary host- plants act as reservoirs for TbYDV which is acquired by the insect during feeding. When primary host-plants become less prevalent the insect moves onto transient hosts such as tobacco and bean, where it transmits TbYDV (Tr ębicki et al. 2010). Outbreaks of the disease are sporadic and the life-cycle of the predominant vector, O. orientalis , on alternate weed hosts, and subsequent acquisition and transmission periods for TbYDV, is poorly understood. Orosius orientalis is mainly controlled through the use of insecticides that kill the insect but do not directly control the disease (Paddick & French 1972). However, the timing and frequency of applications has not been optimised. A better understanding of the tritrophic association between the vector, the virus and its host-plant should
112
provide an opportunity to improve management practices including reduced and optimised insecticide use, introduction of less toxic insecticide alternatives, or perhaps the use of novel protective agents to reduce the incidence of the disease (Tr ębicki et al. 2009). There is currently a paucity of information available on the detection and population dynamics of O. orientalis (Helson 1942; Helson 1951; Osmelak 1987). Therefore, as an important first step towards optimising the management of this leafhopper, we gain insights into the population dynamics and seasonal activity of O. orientalis under field conditions and evaluated three different trapping methods over two cropping seasons.
Materials and methods
Study sites The seasonal activity of O. orientalis was assessed over two growing seasons, 2005-06 and 2006-07, at four field sites (referred to as A-D) on commercial tobacco farms near Myrtleford in the Ovens Valley region of north-east Victoria, Australia. All four sites have been used for tobacco farming for at least two decades (G. Baxter pers. observ.). Sites A and B were neighbouring farms located in the lower Ovens Valley near Whorouly (36°29'23S, 146°35'48E 210 masl), site C was located between Ovens and Eurobin (36°37'12S, 146°48'31E, 246 masl) while site D was located 6 km from Porepunkah (36°39'46S, 146°14'56E, 320 masl).
Sampling methods From December 2005 to June 2006 and September 2006 to June 2007, the relative abundance in traps and seasonal activity of O. orientalis was monitored at all four sites using three different collection methods, namely yellow sticky trap, yellow pan trap and sweep net. Sticky traps and yellow pans were placed in the field, permanently parallel to the tobacco crop (7-8 metre from the crop border), during
113
two collection seasons and examined at weekly intervals to assess leafhopper activity. At each site, 10 sticky traps and 10 pan traps were used. Every 10 m, one sticky trap and one pan trap was placed along a single 100 m transect. Pan traps were placed between the sticky traps but at a distance of 5 m to the right. Sweep nets were used weekly, to assess leafhopper abundance, in the same plot at a position 5 m to the left of pan traps. Transects at sites B, C and D were aligned NW to SE but due to limited area at site A was aligned NE to SW. Prior to conducting this experiment preliminary studies were conducted collecting traps every three hours during the day and once in the 12 hours during the night over several days to determine leafhopper diurnal periodicity and potential interactions between traps. Orosius orientalis was most active in the evening confirming similar observations by Hosking and Danthanarayana (1988) and we did not observe any affects of sweep net on other traps (data not presented). Orosius orientalis when disturbed makes only short rapid flights (Helson 1942) which was estimated as 1 m maximum and therefore a 5 m distance was considered a suitable distance to eliminate between-trap interactions. Despite 5 m distance between traps, prior to collecting using a sweep net, water traps were emptied and sticky traps removed and replaced after sweep net sampling.
Sticky traps Yellow sticky traps (32 cm x 10 cm; Bugs for Bugs, Mundubbera, Australia) were attached to wooden stakes at a height of 20 cm above the plant canopy and were adjusted throughout the season to maintain this height differential. One side of each sticky trap was covered with Tanglefoot®. Using a microspecula and magnifying glass, all leafhoppers were removed from the traps on a weekly basis and transported to the laboratory for counting and identification. Each trap was processed separately and on the same day of collection.
Pan traps Yellow pan or Moericke traps (Moericke 1951; Moericke 1955) were constructed using round, shallow plastic containers (10 x 34 cm diameter) with a pre-moulded groove on the side to enable draining of the collection fluid. All pan traps ware
114
painted yellow using Dulux® spray paint. All pan traps were placed on the ground and filled to 90% capacity with 5-7% saline fixative solution and a few drops of dishwashing detergent to act as a surfactant. The water level in all traps was checked twice between collection dates and topped up if required. The contents of each trap were processed separately by straining the contents through a fine sieve. Insects were gently washed from the sieve using water and were transferred to a screw-top plastic vial containing 70% ethanol prior to segregation and identification. Leafhoppers were counted using a Grale digital cell counter and identified by morphological characteristics using a stereo microscope.
Sweep nets Sweep nets (38 cm diameter, fine mesh) were used by swinging the net through the plant canopy at 180 ° along a 100 m transect where 1 m comprised one sweep. A total of 100 sweeps per site per sampling date were done. The insects collected were placed into a killing jar containing ethyl acetate for 10-15 minutes then transferred into zip-lock plastic bags. To sort samples, four different laboratory test sieves, stacked on top of each other in descending aperture size (3.35 mm, 2 mm, 1 mm and 212 µm), were used to create layers where the sieve with the largest aperture was on the top. The bag contents were emptied to the top sieve and all sieves were shaken for a few seconds. Each sieve was emptied to a white plastic sorting tray and using a magnifying glass and forceps, all leafhoppers were transferred to a Petri dish for identification.
Leafhopper identification Identification of O. orientalis was done using a combination of microscopy and identification keys (Evans 1966; Fletcher 2009; Ghauri 1966). Although O. orientalis was mainly identified by its external characteristics and markings, a detailed examination of the internal structure of male genitalia (Oman 1949) was also carried out where necessary to confirm species.
115
Meteorological data Meteorological data for both collecting seasons were obtained from the Bureau of Meteorology, Wangaratta, Victoria and SILO database (http://www.bom.gov.au/silo/).
Statistical analysis
Statistical analysis was conducted using Genstat software (10th edn 2007, Lawes Agricultural Trust). A one-way analysis of variance (ANOVA) was used to compare differences between the numbers of O. orientalis trapped using yellow sticky traps and yellow pan traps from September 2006 to February 2007.
Results
Seasonal activity A total of over 28,000 individual O. orientalis were caught from vegetation adjacent to tobacco fields at the four sites (A, B, C and D) using the three different collection methods during two collecting seasons (2005-06 and 2006-07). The total number of O. orientalis caught during the 2005-06 tobacco season (December-May) was three-fold lower (n=7046) than that caught in season 2006- 07 (September-May; n = 21867) (Table 5.1). Climatic conditions differed between seasons with higher annual rainfall in the 2005-06 season compared to 2006-07 (Table 5.2).
116
Table 5.1 Number of Orosius orientalis recorded from two seasons (05/06 and 06/07) and four sites (A, B, C and D) using three different trapping methods (sweep net, sticky trap and pan trap).
Season 2005-2006 2006-2007 Site Trap type Trap type Sticky Sticky Sweep net Pan trap Sweep net Pan trap trap trap A 81 272 972 114 561 1969 B 403 456 1250 594 1202 3573 C 103 321 2869 1119 1176 8048 D 25 72 222 401 539 2571
Total 612 1121 5313 2228 3478 16161
117
Table 5.2 Average minimum and maximum temperature and total annual rainfall recorded at the four field sites (A–D) over 3 years.
Year 2005 2006 2007 Temperature (°C) Temperature (°C) Temperature (°C) Sites Maximum Minimum Rain (mm) Maximum Minimum Rain (mm) Maximum Minimum Rain (mm) A & B 21.71 7.63 815.4 23.15 7.1 333.4 22.98 8.88 653.7 C 21.32 7.82 1229.8 22.66 7.44 449.4 22.52 9.07 954.8 D 18.92 6.72 1196.8 20.22 6.47 476.8 20.04 7.86 863.5
118
Trap evaluation From all four sampling sites during both seasons, the yellow pan traps caught a four-fold and over seven-fold greater number of O. orientalis , respectively, than sticky traps and the sweep net (Table 5.1; Figure 5.1). Furthermore, statistical analyses revealed that there was a significant difference (P>0.05) between yellow pan trap and yellow sticky trap data from all sites for each season. Since sweep net sampling was a one-off weekly event, rather than a cumulative weekly sampling event as used with yellow pan and sticky traps, this data was not compared statistically and any comparisons in relative abundance for sampling method should be treated with caution.
Leafhopper population dynamics Irrespective of trap type, a trimodal peak in O. orientalis in activity was generally observed at all four sites (Figure 5.2). However, the relative activity of O. orientalis differed between seasons and sites, with a relatively higher activity in the 2006-07 season which was characterised by relatively low annual rainfall and warmer temperatures (Table 5.2). The first population peak occurred early in the season around mid-September (early spring), the second in late November and the third in early January (summer). For the 2006-07 season, the highest activity of O. orientalis at sites C and D occurred in the spring, whereas at sites A and B the peak was recorded during the summer. From the end of January till late May, relatively low numbers of O. orientalis were trapped at all sites.
119
Figure 5.1 Comparison of catches of Orosius orientalis using yellow sticky traps and yellow pan traps from September 2006 to February 2007 from field sites A-D [LSD = 3.64, * P = 0,05].
120
Figure 5.2 Population dynamics of Orosius orientalis over two seasons using three trapping methods at field sites A-D.
121
Discussion
Although the population dynamics of Orosius orientalis have been examined previously under Australian conditions, most of these studies have focused on the association of the leafhopper with lucerne ( Medicago sativa L.) (Helson 1951; Pilkington et al. 2004b) or tomato ( Lycopersicon esculentum Mill) (Osmelak 1987) and most were carried out over a single cropping season using predominantly light traps and suction traps. In this study, we examined the population dynamics of O. orientalis associated with four different tobacco- growing field sites over two seasons using three trapping methods: yellow sticky traps, yellow pan traps and sweep nets. Orosius orientalis was recorded at all four sites, with peak activity occurring in the spring and summer months. Furthermore, yellow pan traps consistently caught significantly higher numbers of O. orientalis than yellow sticky traps. Previous studies on the population dynamics of O. orientalis outside crop areas indicated that the most suitable leafhopper trapping methods were incandescent light traps and suction traps, with water baffle (similar to yellow pan traps), ultra violet light and pitfall traps less suitable (Osmelak 1987; Osmelak & Fletcher 1988). Suction traps have also been used successfully to monitor low level flight activity of O. orientalis in grasslands (Hosking & Danthanarayana 1988), whereas studies conducted in sesame fields in Turkey showed yellow sticky traps were more effective than suction traps (Kersting et al. 1997). Trap height has also been found to be an important consideration when trapping O. orientalis (Pilkington et al. 2004a). Although incandescent light, vacuum sampling (Holtkamp & Thompson 1985) and mouth aspirator collection methods were also trialled during the first two months of our monitoring period, these were discontinued in favour of sticky traps, pan traps and sweep nets due to the relatively low catch numbers (data not presented). In contrast to previous studies, we identified three peaks of activity of O. orientalis at all four sites. The first population peak occurred in early spring (mid-September), the second in late November and the third in summer (early January). The summer sampling period is particularly important as this is when TbYDV has been shown to be present in
122
both O. orientalis and weed species surrounding tobacco fields (Tr ębicki et al. 2010). In a single tobacco growing season in the Ovens Valley, three generations of O. orientalis have been previously observed, first in mid-December, a second in February and a third in mid-March (Helson 1942). Prior to our study, there was limited published data comparing the population dynamics of O. orientalis at different sites over successive seasons. Using five different trapping methods over a single season, regular peaks of O. orientalis activity have previously been observed in tomato crops from mid-November till February (Osmelak 1987). In a single season study in lucerne, maximum activity of O. orientalis , assessed using sweep net catches, were reported in mid-November with high levels till the end of December and smaller peaks in late February and mid-September (Helson 1951). The differences between this study and research conducted previously may be explained by trapping protocol and design as well as climatic conditions. During our collecting seasons, for example, sites experienced conditions of very low rainfall and high temperature. Between-site differences in O. orientalis activity have been reported which are most likely to have been influenced by climate variability and/or vegetation type and diversity (Tr ębicki et al. 2010). Regardless of these differences, however, the yellow pan trap consistently trapped more O. orientalis than either both sticky trap and sweep net. The identification of leafhopper activity peaks is fundamentally important information required to optimise chemical control options for O. orientalis and the pathogens it vectors such as TbYDV. It is also important to determine when and by which method, to detect sufficient O. orientalis to conduct disease vector studies (Tr ębicki et al. 2010). In summary, yellow pan traps were found to be the most effective method for trapping O. orientalis and characterising population dynamics in this study. However, for practicality, it may be more prudent to use less time consuming approaches in peak periods of O. orientalis activity, such as sweep nets which do give a good indication of leafhopper abundance and are relatively simple to use, particularly for vector studies where the requirement is to collect fresh insect samples for virus testing. The results of this study have provide important information which can be used to develop more effective disease control strategies for O. orientalis not only in Australia but also worldwide. Orosius orientalis transmits a range of
123
economically important viruses and phytoplasmas in a variety of crops (Harding & Teakle, 1985; Helson, 1951; Hutton & Grylls, 1956; Kersting et al. 1997; Osmelak, 1986). For example the seasonal activity data from this study when linked to pathogen studies will help determine the most appropriate times for insecticide applications lead to improved control strategies for this economically important vector.
Acknowledgements
The assistance of Jack Harrington, Anna Harrington, Giuseppe Sacco, growers from NE Victoria, colleagues from DPI Rutherglen and all comments from two anonymous reviewers is gratefully acknowledged. This research was funded by Horticulture Australia Limited and the Tobacco Research and Development Corporation with in-kind support from the Department of Primary Industries, Victoria, Australia.
References
Day MF & Fletcher MJ. 1994. An annotated catalogue of the Australian Cicadelloidea (Hemiptera: Auchenorrhyncha). Invertebrate Taxonomy 8, 1117- 1288.
Dietrich CH. 2005. Keys to the families of Cicadomorpha and subfamilies and tribes of Cicadellidae (Hemiptera: Auchenorrhyncha). Florida Entomologist 88 , 507-517.
Evans JW. 1966. The leafhoppers and froghoppers of Australia and New Zealand (Homoptera: Cicadelloidea and Cercopoidea). The Australian Museum Memoir XII , 1-348.
124
Fletcher MJ. 2000. Identification keys and checklists for the leafhoppers, planthoppers and their relatives occurring in Australia and New Zealand (Hemiptera: Auchenorrhyncha). http://www.agric.nsw.gov.au/Hort/ascu/start.htm Orange Agricultural Institute, NSW DPI, Orange, New South Wales .
Ghauri MSK. 1966. Revision of the genus Orosius Distant (Homoptera: Cicadelloidea). Bulletin of the British Museum (Natural History), Entomology 18 , 231-252.
Grylls NE. 1979. Leafhopper vectors in Australia. In Leafhopper Vectors and Plant Disease Agents (Ed. K Maramorosch and K F Harris.). Academic Press New York , 179-214.
Harding RM & Teakle DS. 1985. Mycoplasma-like organisms as causal agents of potato purple top wilt in Queensland. Australian Journal of Agricultural Research 36 , 443-449.
Helson GAH. 1942. The leafhopper Thamnotettix argentata Evans, a vector of tobacco yellow dwarf. Journal of Council of Scientific and Industrial Research 15 , 175-184.
Helson GAH. 1951. The transmission of witches broom virus disease of lucerne by the common brown leafhopper, Orosius argentatus (Evans). Australian Journal of Scientific Research, Series B - Biological Science 4, 115-124.
Hill AV. 1941. Yellow dwarf of tobacco in Australia, II. Transmission by the jassid Thamnotettix argentata (Evans). Journal of the Council for Scientific and Industrial Research 14 , 181-186.
Hill AV. 1950. Yellow dwarf of tobacco in Australia, IV. Some host-plants of the virus. Australian Journal of Agricultural Research 1, 141-143.
Holtkamp RH & Thompson JI. 1985. A lightweight, self-contained insect suction sampler. Journal of the Australian Entomological Society 24 , 301-302.
125
Hosking JR & Danthanarayana W. 1988. Low level flight activity of Nesoclutha pallida (Evans), Orosius argentatus (Evans) and Zygina zealandica (Myers) (Hemiptera: Cicadellidae) in Southern Victoria. Journal of the Australian Entomological Society 27 , 241-249.
Hutton EM & Grylls NE. 1956. Legume 'little leaf', a virus disease of subtropical pasture species. Australian Journal of Agricultural Research 7, 85-97.
Kersting U, Baspinar H, Uygun N & Satar S. 1997. Comparison of two sampling methods for leafhoppers (Homoptera, Cicadellidae) associated with sesame in the east Mediterranean region of Turkey. An. Schadlingskde., Pflanzenschutz, Umweltschutz 70 , 131-135.
Moericke V. 1951. Eine Farbfalle zur Kontrolle des Fluges von Blattlausen, insbesondere der Pfirsichblattlaus Myzodes persicae (Sulz). Nachrichtenblatt des Deutschen Pflanzenschutzdienste. Stuttgart 3, 23-24.
Moericke V. 1955. Über die Lebensgewohnheiten der geflügelten Blattläuse (Aphidina) unter besonderer Berücksichtigung des Verhaltens beim Landen. Zeitschrift für Angewandte Entomologie 37 , 29-91.
Moran JR & Rodoni B. 1999. Strategies for the control of tobacco yellow dwarf virus. Report on a consultancy conducted on behalf of the Tobacco Corporation of Victoria. 1-5.
Oman PW. 1949. The Nearctic leafhoppers (Homoptera: Cicadellidae): a generic classification and check list. Memoirs of the Entomological Society of Washington 3, 1-253.
Oman PW, Knight WJ & Nielson MW. 1990. Leafhoppers (Cicadellidae). A bibliography, generic check-list and index to the world literature 1956-1985 , CAB International Institute of Entomology, Wallingford.
Osmelak JA. 1987. A comparison of five different traps for monitoring leafhopper activity (Homoptera). General and Applied Entomology 19 , 52-56.
126
Osmelak JA & Fletcher MJ. 1988. Auchenorrhyncha (Hemiptera: Cicadellidae, Fulgoroidea, Cercopidae) trapped near tomato crops at Tatura, Victoria. General and Applied Entomology 20 , 52-56.
Paddick RG & French FL. 1972. Suppression of tobacco yellow dwarf with systematic organophosphorus insecticide. Australian Journal of Experimental Agriculture and Animal Husbandry 12 , 331-334.
Pilkington LJ, Gurr GM, Fletcher MJ, Elliott E, Nikandrow A & Nicol HI. 2004a. Reducing the immigration of suspected leafhopper vectors and severity of Australian lucerne yellows disease Australian Journal of Experimental Agriculture 44 , 983–992
Pilkington LJ, Gurr GM, Fletcher MJ, Nikandrow A & Elliott E. 2004b. Vector status of three leafhopper species for Australian lucerne yellows phytoplasma. Australian Journal of Entomology 42, 366-373.
Thomas JE & Bowyer JW. 1980. Properties of tobacco yellow dwarf and bean summer death viruses. Phytopathology 70 , 214-217.
Tr ębicki P, Harding RM & Powell KS. 2009. Antimetabolic effects of Galanthus nivalis agglutinin and wheat germ agglutinin on nymphal stages of the common brown leafhopper using a novel artificial diet system. Entomologia Experimentalis et Applicata 131 , 99-105.
Tr ębicki P, Harding RM, Rodoni B, Baxter G & Powell KS. 2010. Vectors and alternative hosts of TbYDV in south-eastern Australia. Annals of Applied Biology 157 , 13-24.
Tr ębicki P, Powell KS & Baxter G. 2007. Epidemiology and management of leafhopper that transmit a unique virus In: Sustainable tobacco production (eds G Baxter, G Hayes, A Harrington, J Harrington & G Sacco) pp.132. Department of Primary Industries, Final Report, TBO4001.
127
van Rijswijk B, Rodoni BC, Baxter G & Moran JR. 2002. Managing tobacco yellow dwarf virus. Phase 1: Developing a diagnostic tool. Phase 2: Beginning to understand the epidemiology of TYDV. In: Department of Natural Resources and Environment pp.48. Tobacco Research and Development Corporation Final Report 1999/2001 of Project DAV 35T.
128
CHAPTER 6
ANTIMETABOLIC EFFECTS OF GALANTHUS NIVALIS AGGLUTININ AND WHEAT GERM AGGLUTININ ON NYMPHAL STAGES OF THE COMMON BROWN LEAFHOPPER USING A NOVEL ARTIFICIAL DIET SYSTEM
Piotr Tr ębicki 1, Rob M. Harding 1 & Kevin S. Powell 2
1Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Queensland, GPO Box 2434, Brisbane, 4001, Australia, and
2Department of Primary Industries, Biosciences Research Division, Rutherglen, Victoria, 3685, Australia
* Current address, Kevin S. Powell, Department of Primary Industries, Biosciences Research Division, RMB 1145, Chiltern Valley Road, Rutherglen,
Entomologia Experimentalis et Applicata ( 2009), 131 (1), 99-105.
129
STATEMENT OF AUTHORSHIP
Piotr Tr ębicki (principal author): Executed the work (designed and conducted all laboratory experiments, analysed and interpreted data) and wrote initial manuscript.
Signed ……………………………………. Date …18-12-09..…………………..
Rob Harding: Conceived project idea, critically interpreted data and contributed to final manuscript.
Signed ……………………………………. Date …18-12-09…...………………..
Kevin Powell: Conceived project idea, supervised the work, critically interpreted data and contributed to final manuscript.
Signed ……………………………………. Date …18-12-09...…………………..
130
Abstract
A chemically defined artificial diet was developed to screen potential control agents for the common brown leafhopper, Orosius orientalis (Matsumura) (Hemiptera: Cicadellidae), a known vector of several economically important phytoplasmas and plant viruses. In vitro feeding trials were carried out to optimise insect survival and development on two artificial diets. A novel diet formulation allowed survival for O. orientalis up to 46 days and full development from first instar through to adulthood. The effect of three selected plant derived proteins, cowpea trypsin inhibitor (CpTi), Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA) on survival and development was assessed using the in vitro protocol. GNA and WGA reduced survival and development significantly when incorporated at 0.1% (w/v) concentration. In contrast CpTi at the same concentration did not show significant antimetabolic properties. Based on these results, GNA and WGA are potentially useful antimetabolic agents for expression in genetically modified crops to improve the management of O. orientalis and the pathogens it vectors.
131
Introduction
The common brown leafhopper, Orosius orientalis (Matsumura) (Homoptera: Cicadellidae), previously described as Orosius argentatus (Evans), is an important vector of several viruses and phytoplasmas worldwide. In Australia, phytoplasmas vectored by O. orientalis cause a range of economically important diseases, including legume little leaf (Hutton & Grylls 1956), tomato big bud (Osmelak 1986), lucerne witches broom (Helson 1951), potato purple top wilt (Harding & Teakle 1985), and Australian lucerne yellows (Pilkington et al. 2004). Orosius orientalis also transmits Tobacco yellow dwarf virus (TYDV; genus Mastrevirus, family Geminiviridae) to beans, causing bean summer death disease (Ballantyne 1968), and to tobacco, causing tobacco yellow dwarf disease (Hill 1937; 1941). TYDV has only been recorded in Australia to date. Both diseases result in significant production and quality losses (Ballantyne 1968; Moran & Rodoni 1999; Thomas 1979). Although direct damage caused by leafhopper feeding has been observed, it is relatively minor compared to the losses resulting from disease (P Tr ębicki, unpubl.). Control strategies for O. orientalis are primarily based on the use of chemical insecticides (Osmelak 1986; Paddick & French 1972; Paddick et al. 1971). However, due to the paucity of information available on the life-cycle, population dynamics, and disease transmission characteristics of this leafhopper, the use of insecticides has been largely ineffective. An alternative approach to controlling sap-sucking insect pests is the use of antimetabolites, such as plant lectins expressed in genetically modified plants (Gatehouse et al. 1992; Peumans & Van Damme 1995). Using artificial diet bioassay systems, a wide range of plant lectins, including Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA), have been shown to exhibit antimetabolic effects against many economically important Homopteran pests resulting in reduced survival and delayed development (Chen 2008; Gatehouse et al. 1995; Habibi et al. 1993; Powell et al. 1993). Furthermore, transgenic plants expressing lectins have shown enhanced resistance towards many sap-sucking insects, including the rice brown planthopper, Nilaparvata lugens (Stål) and green leafhopper, Nephotettix spp.
132
(Gatehouse & Gatehouse 1998; Jouanin et al. 1998; Peferoen 1997). Based on these studies, the use of antimetabolites may also potentially be an effective strategy to control O. orientalis . A prerequisite to preliminary screening of potential antimetabolites in vitro is the development of a chemically defined artificial diet for the target pest. The basic nutritional requirements of O. orientalis are poorly understood and, although artificial diets for several Cicadellidae have been developed (Cohen 2004; Singh 1977), a diet or diet feeding system has not been reported for O. orientalis . In this study, we describe the development of the first artificial diet bioassay system for rearing O. orientalis in vitro and a simple oviposition chamber for collection of newly emerged first instars. In addition, we describe the use of this diet bioassay system to assess the antimetabolic effects of two plant lectins and a protease inhibitor towards first instars of the leafhopper.
Materials and methods
Insect culture Stock colonies of O. orientalis were obtained from Charles Darwin University, Darwin, Australia, and from herbaceous vegetation surrounding commercial tobacco farms in the Ovens Valley, NE Victoria, Australia (36°37'12S, 146°48'31E). Leafhoppers were identified to species level using morphological characteristics of the male genitalia (Evans 1966; Fletcher 2000; Ghauri 1966). Cultures were maintained, on 30–40-day-old celery [ Apium graveolens L. (Apiaceae)] or bean [ Phaseolus vulgaris L. (Fabaceae)] plants grown under glasshouse conditions, for eight generations before fecund female adults were transferred to oviposition chambers and reared under controlled environment conditions (25 ± 2 °C, L14:D10). The oviposition chambers (Figure 6.1) were made by modifying standard plastic Petri dishes (9 × 2.5 cm). Three access holes on opposite sides of the dish base were made. A 3-4 cm diameter opening was made in the lid and sealed with glued mesh to prevent first-instar escape, facilitate air circulation, and reduce condensation. One attached leaf of the host-plant was
133
placed in the dish base with the stem inserted into the access hole, covered with the lid and sealed with an elastic band. The two remaining access holes allowed introduction of fecund adult females and removal of newly emerged first instars. To prevent insect escape during oviposition or post hatching, foam was wrapped around the stem of the leaf and two foam stoppers were placed into each access hole. Orosius orientalis oviposits on the leaf blade, petiole, or stem (Helson 1942), and using this chamber design fecund females could freely choose oviposition sites.
Chemicals and materials Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA) were obtained from Abacus Australian Laboratory Supplies, East Brisbane, Australia. DL-homoserine was purchased from MP Biomedicals Australasia, Seven Hills, Australia. Cowpea trypsin inhibitor (CpTi) and all remaining dietary components were obtained from Sigma-Aldrich, Castle Hill, Australia. All chemicals utilized in artificial diet preparation had the highest purity commercially available.
Artificial diet preparation Two artificial diets were prepared for rearing nymphal stages of O. orientalis , MED-1 as previously utilised for the smaller brown leafhopper, Laodelphax striatellus Fallen (Mitsuhashi 1974), and a modification of MED-1 designated PT- 07. Dietary modifications were made to the amino acid and vitamin profile and concentration, and by the addition of cholesteryl benzoate (Table 6.1). All components were dissolved in sterile ultra pure water using gentle heat (25°C), and the pH was adjusted to 6.5 with 1 M potassium hydroxide. After filtration through a 0.2-µm Millipore disposable filter, diet solutions were dispensed into 50-ml plastic containers as stock solutions, and further dispensed into 1.5-ml Eppendorf tubes as working solutions and stored at -20 °C prior to use.
134
Figure 6.1 Oviposition chamber for Orosius orientalis on (A) host-plant with (B) rubber band closure, (C) fabric mesh ventilation point, (D) Petri dish, (E) access points for nymph removal and adult addition, foam plugs to prevent insect escape and (F) leaf attached to whole bean or celery host-plant.
135
136
Table 6.1 Composition (mg/l) of artificial diets, PT-07 and MED-11, used for rearing Orosius orientalis .
Ingredient PT-07 MED-1 Ingredient PT-07 MED-1
L-alanine 1 000 1 000 MgCl 2·6H 2O 2 000 2 000
γ-amino butyric acid 200 200 KH 2PO 4 5 000 5 000
L-arginine hydrochloride 3 000 4 000 CaCl 2·2H 2O 32 31.15
L-asparagine 4 000 3 000 CuCl 2·2H 2O 3 2.68
L-aspartic acid 1 000 1 000 FeCl 3·6H 2O 23 22.28
L-cysteine 500 500 MnCl 2·4H 2O 8 7.93
L-cystine hydrochloride 50 ZnCl 2 5 3.96 L-glutamic acid 1 500 2 000 L-glutamine 6 000 6 000 Biotin 1 1 Glycine 400 200 Calcium pantothenate 50 50 L-histidine 1 500 2 000 Choline chloride 500 500 DL-homoserine 8 000 Folic acid 10 10 L-isoleucine 1 500 2 000 Inositol 500 500 L-leucine 1 500 2 000 Nicotinic acid 100 100 L-lysine hydrochloride 1 800 2 000 Pyridoxine hydrochloride 25 25 L-methionine 1 500 1 000 Riboflavin 25 50 L-phenylalanine 1 000 1 000 Thiamine hydrochloride 25 25 L-proline 1 000 1 000 Ascorbic acid 1 000 L-serine 1 000 1 000 Sodium ascorbate 1 000 L-threonine 1 500 2 000 Cholesteryl benzoate 25 L-tryptophane 1 000 1 000 L-tyrosine 200 200 Sucrose 50 000 50 000 L-valine 1 500 2 000
pH of both diets adjusted with KOH to 6.5, 1Source: Mitsuhashi, 1974.
137
Feeding trials The effect of artificial diets, MED-1, and PT-07 on the development and survival of O. orientalis was examined using feeding chambers essentially as described by Powell et al . (1993) but with a minor modification. The feeding chamber was placed in a second, larger plastic Petri dish (9 × 2.5 cm) containing wet filter paper to increase and maintain constant humidity (Figure 6.2). Five newly emerged first instars of O. orientalis were removed from oviposition chambers with a fine wet paint brush and placed in the feeding chamber (plastic Petri dish, 1 × 3.5 cm). A single layer of stretched Parafilm M was placed over the chamber and 100 µl of diet was deposited on the membrane. A second layer of Parafilm M was then stretched over the artificial diet to form a feeding sachet. Two controls (no diet and water only) were included in all experiments and 10 replicates were used for each treatment and control. To examine the effect of antimetabolites on the survival of O. orientalis , GNA, WGA, and CpTi were separately incorporated into the PT-07 artificial diet at a concentration of 0.1% (wt/vol). Five newly emerged first instars of O. orientalis were used in each feeding chamber as described above. ‘No diet’ and PT-07 diet without the inclusion of a plant protein, were included as controls. Ten replicates were used for each treatment and the control. In all feeding trials, leafhopper survival data was recorded daily and diets were replaced on alternative days. To avoid insect escape during the diet changing procedure, insects were temporarily immobilised by placing the feeding chamber at -20 °C for 90 s. To avoid fungal growth on excreted honeydew, feeding chambers were replaced weekly under aseptic conditions. Parafilm M and feeding chambers were exposed to ultraviolet light for 20 min prior to insect introduction. All trials were conducted in controlled growth rooms (25 ± 2 °C, L14:D10).
138
Figure 6.2 Feeding chamber for rearing Orosius orientalis on liquid diet through a double layer of Parafilm M .
139
140
Statistical analysis Statistical analysis was performed using GenStat software (10th ed. © 2007, Lawes Agricultural Trust). A Kaplan-Meier estimate and log-rank test was used to determine survivor distribution and to compare differences between diet formulations and diets with antimetabolic compounds. Corrected mortality calculations (Abbott 1925) were used to compare the relative efficacy of treatments.
Results and discussion
Modifications were made to the MED-1 (Mitsuhashi 1974) diet formulation resulting in the development of a novel artificial diet PT-07. These modifications included changing the proportions of amino acids (L-arginine hydrochloride, L- asparagine, L-glutamic acid, glycine, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-threonine, and L-valine), inorganic salts, and including cholesteryl benzoate and ascorbic acid, which resulted in enhanced survival and development of O. orientalis. PT-07 artificial diet was significantly more effective for rearing O. orientalis than the MED-1 diet (Figure 6.3) increasing development and survival. In the absence of diet, using either no-diet or water-only controls, insects survived for a maximum of 5 days. In contrast, the survival of O. orientalis on the PT-07 diet was significantly enhanced compared to the MED-1 diet and the controls (log- rank test P<0.001). Insects reared on the MED-1 diet survived for a maximum of 14 days whereas the PT-07 diet sustained insects for up to 46 days reaching adulthood at day 42-45. When 100% mortality of leafhoppers was reached on MED-1 diet, more than 80% of insects remained alive on the PT-07 diet. Insects reared on MED-1 did not develop beyond the third instar (with a large proportion not surviving ecdysis (data not presented), whereas PT-07 supported leafhopper development to the adult stage. On host-plants leafhopper survival and development varies depending on plant species as this insect is polyphagous with up to 20 confirmed host-plant species (P Tr ębicki, unpubl.). Under laboratory
141
conditions on average it takes 25 days till nymphs reach adulthood when rearing on Malva parviflora L. (Helson 1942) and up to 35 days on celery plant (P Tr ębicki, unpubl.). Liquid artificial diets for rearing homopteran pests have been used for decades (Vanderzant 1974) along with a feeding sachet system for delivery which was first developed for leafhoppers (Carter 1927). Artificial diets, which enable the rearing of Cicadellidae and Delphacidae through successive generations (Mitsuhashi 1974; Mitsuhashi & Koyama 1972), have been developed but none prior to this study was available for O. orientalis . Optimization of the concentration and proportion of amino acids in a diet is an important factor for leafhopper development and survival as they are known to have phagostimulatory and phagoinhibitory properties (Sogawa 1977). Some leafhoppers also require a source of cholesterol for optimal development (Lin & Hou 1981). A number of amino acids, including L-Asparagine, were proven to act as a sucking stimulant for N. lugens (Sogawa 1972) and the increased concentration of this amino acid may have improved survival of O. orientalis on PT-07 diet.
142
Figure 6.3 The effect of two artificial diet formulations, MED-1 and PT-07, on the survival and development of first instars of Orosius orientalis . Each data point represents the mean of 10 replicates, each of which contained 5 insects at the commencement of the experiment.
143
First instars of O. orientalis were also exposed to the PT-07 diet containing 0.1% (wt/vol) concentrations of either GNA, WGA, or CpTi, with PT-07 diet only and no diet used as a control. Using the PT-07 control only diet, some insects were still alive after 43 days. Cowpea trypsin inhibitor (CpTi) showed no significant effects on leafhopper survival or development (Figure 6.4) with a corrected mortality of only 4%. In contrast, GNA and WGA both showed significant antimetabolic effects (log-rank test P<0.001), with nymphal survival reduced to 22 and 15 days, respectively. Although the corrected mortality values using WGA and GNA were relatively high at 37 and 35%, respectivel and significantly different to CpTi treatment (log-rank test P<0.001), they were not statistically different from one another (P>0.1). These mortality levels, although lower than those reported using BPH with N. lugens (up to 76%) (Powell et al. 1993), were nonetheless significant. The comparatively broad host-plant range of O. orientalis (Helson 1942) may partially explain the difference in mortality compared to N. lugens which is monophagus on rice . Other studies have shown that more GNA binds to N. lugens gut tissue compared to corresponding tissue of the rice green leafhopper (GLH), Nephotettix spp., despite the fact that GLH ingested more plant sap from GNA-transformed rice plants (Foissac et al. 2000).
144
Figure 6.4 The effect of Galanthus nivalis agglutinin (GNA), wheat germ agglutinin (WGA) and cowpea trypsin inhibitor (CpTi) when incorporated at 0.1% (wt/vol) in artificial diet PT-07 on the survival of first instar nymphs of Orosius orientalis . Each data point represents the mean of ten replicates, each of which contained five insects at the commencement of the experiment.
145
Previous studies, using either an in vitro artificial diet bioassay system for screening antimetabolites or by expressing proteins in transgenic plants, have shown that a number of plant-derived compounds affect the development of a range of Homoptera. GNA and WGA, for example, have shown significant antimetabolic effects towards the rice green leafhopper (GLH, Nephotettix cinciteps Uhler) and rice brown planthopper (BPH, N. lugens ) (Foissac et al. 2000; Powell et al. 1993; 1995a; Powell et al. 1995b). Immunolabelling studies have shown that GNA binds to cell carbohydrate moieties in the gut of BPH causing a granular appearance of the midgut epithelial cells, as evidenced by disruption of the microvilli (Powell et al. 1998). GNA has also been shown to be an effective antimetabolic agent for several aphid species (Sauvion et al. 1996) causing delayed development and reduced survival. In this study, CpTi showed no significant effects on leafhopper survival or development. Previous studies have shown that, although CpTi incorporated into artificial diets or expressed in transgenic plants reduce survival in some lepidopteran (Bell et al. 2001; Hilder et al. 1987), coleopteran (Graham et al. 1997), and orthopteran (Boulter et al. 1989) species, this protein was not considered effective enough to be regarded as a viable control agent for Homoptera (Boulter et al. 1989). Similar observations were obtained from N. lugens and N. cinciteps fed on artificial diets containing CpTi (Powell et al. 1993). In our study, two plant lectins, GNA and WGA, have been identified as potential control agents for O. orientalis . However, further in vitro and in planta studies are required to determine the effectiveness of this approach in transgenic crops. The mechanism of action of these lectins towards O. orientalis also requires further investigation as this could impact on its effectiveness as a vector. Both GNA and WGA have been shown to have an antifeedant effect against planthoppers (Powell et al. 1995b) resulting in increased probing activity (Powell & Gatehouse 1996) and both lectins also bind to insect midgut epithelial cells (Eisemann et al. 1994; Powell et al. 1998) and this is dependant on the binding site affinity with GNA and WGA binding to D-mannose and N-acetyl glucosamine sites, respectively (Sharon & Lis 1989). In previous studies, several crop species including tobacco have been genetically modified to express GNA and this approach has led to reduced survival of aphids and planthoppers (Chen 2008; Hilder et al. 1995; Rao et al.
146
1998; Stoger et al. 1999). Since current chemical control agents for O. orientalis have little effect in reducing the incidence of this leafhopper and the diseases it transmits, expressing lectins with different modes of action in transgenic plants through gene pyramiding (Burrows et al. 1999) may be an alternative strategy to provide more effective control and to combat the development of potential resistance-breaking genotypes. However, selection of an appropriate promoter gene could also influence the epidemiology of disease transmission. A comparison between constitutive promoters or phloem specific promoters (Wang et al. 2005) would be advisable for O. orientalis as TYDV is phloem restricted (Needham et al. 1998).
Acknowledgements
This research was funded by Horticulture Australia Limited and the Tobacco Research and Development Corporation with in-kind support from the Department of Primary Industries (DPI), Victoria, Australia. The assistance of Brendan Rodoni (DPI Knoxfield), Gary Baxter (DPI Ovens), and Lucy Tran- Nguyen (Charles Darwin University, Australia), is gratefully acknowledged.
References
Abbott WS. 1925. A method of computing the effectiveness of an insecticide. Journal of Economic Entomology 18 , 265-267.
Ballantyne B. 1968. Summer death of beans. Agricultural Gazette of New South Wales 79 , 486-489.
Bell HA, Fitches EC, Down RE, Ford L, Marris GC, Edwards JP, Gatehouse JA & Gatehouse AMR. 2001. Effect of dietary cowpea trypsin inhibitor (CpTi) on the growth and development of the tomato moth Lacanobia oleracea (Lepidoptera:
147
Noctuidae) and on the success of the gregarious ectoparasitoid Eulophus pennicornis (Hymenoptera: Eulophidae). Pest Management Science 57 , 57-65.
Boulter D, Gatehouse AMR & Hilder VA. 1989. Use of cowpea trypsin inhibitor (CpTi) to protect plants against insect predation. Biotechnology Advances 7, 489 - 497.
Burrows PR, Barker ADP, Newell CA & Hamilton WDO. 1999. Plant-derived enzyme inhibitors and lectins for resistance against plant-parasitic nematodes in transgenic crops. Pesticide Science 52 , 176-183.
Carter W. 1927. A technique for use with Homopterous vectors of plant diseases, with special reference to the sugar beet leafhopper, Eutettix tenallus (Baker). Journal of Agricultural Research 34 , 449-451.
Chen M-S. 2008. Inducible direct plant defence against insect herbivores: A review. Insect Science 15 , 101-114.
Cohen AC. 2004. Insect diets. Science and Technology , CRC Press LLC, Boca Raton.
Eisemann CH, Donaldson RA, Pearson RD, Cadagon LC, Vuocolo T & Tellam RL. 1994. Larvicidal activity of lectins on Lucilia cuprina : Mechanism of action. Entomologia Experimentalis et Applicata 72 , 1-11.
Evans JW. 1966. The leafhoppers and froghoppers of Australia and New Zealand (Homoptera: Cicadelloidea and Cercopoidea). The Australian Museum Memoir XII , 1-348.
Fletcher MJ. 2000. Identification keys and checklists for the leafhoppers, planthoppers and their relatives occurring in Australia and New Zealand (Hemiptera: Auchenorrhyncha). http://www.agric.nsw.gov.au/Hort/ascu/start.htm Orange Agricultural Institute, NSW DPI, Orange, New South Wales .
148
Foissac X, Thi Loc N, Christou P, Gatehouse AMR & Gatehouse JA. 2000. Resistance to green leafhopper ( Nephotettix virescens ) and brown planthopper (Nilaparvata lugens ) in transgenic rice expressing snowdrop lectin ( Galanthus nivalis agglutinin; GNA). Journal of Insect Physiology 46 , 573-583.
Gatehouse AMR, Boulter D & Hilder VA. 1992. Potential of plant-derived genes in the genetic manipulation of crops for insect resistance. In: Plant Genetic Manipulation for Crop Protection (eds AMR Gatehouse, D Boulter & VA Hilder) 135-153. CAB International, Wallingford, United Kingdom.
Gatehouse AMR & Gatehouse JA. 1998. Identifying proteins with insecticidal activity: use of encoding genes to produce insect-resistant transgenic crops. Pesticide Science 52 , 165-175.
Gatehouse AMR, Powell KS, Peumans WJ, Van Damme EJM & Gatehouse JA. 1995. Insecticidal properties of plant lectins: their potential in plant protection. In: Lectins: Biomedical Perspectives (eds AJ Pusztai & S Bardocz) 35-57. Taylor & Francis, London.
Ghauri MSK. 1966. Revision of the genus Orosius Distant (Homoptera: Cicadelloidea). Bulletin of the British Museum (Natural History), Entomology 18 , 231-252.
Graham J, Gordon SC & McNicol RJ. 1997. The effect of the CpTi gene in strawberry against attack by vine weevil ( Otiorhynchus sulcatus F. Coleoptera: Curculionidae). Annals of Applied Biology 131 , 133-139.
Habibi J, Backus EA & Czapla TH. 1993. Plant lectins affect survival of the potato leafhopper (Homoptera: Cicadellidae). Journal of Economic Entomology 86 , 945-951.
Harding RM & Teakle DS. 1985. Mycoplasma-like organisms as causal agents of potato purple top wilt in Queensland. Australian Journal of Agricultural Research 36 , 443-449.
149
Helson GAH. 1942. The leafhopper Thamnotettix argentata Evans, a vector of tobacco yellow dwarf. Journal of Council of Scientific and Industrial Research 15 , 175-184.
Helson GAH. 1951. The transmission of witches broom virus disease of lucerne by the common brown leafhopper, Orosius argentatus (Evans). Australian Journal of Scientific Research, Series B - Biological Science 4, 115-124.
Hilder VA, Gatehouse AMR, Sheerman SE, Barker RF & Boulter D. 1987. A novel mechanism of insect resistance engineered into tobacco. Nature 330 , 160- 163.
Hilder VA, Powell KS, Gatehouse AMR, Gatehouse JA, Gatehouse LN, Shi Y. Hamilton WDO, Merryweather A, Newell CA, Timans JC, Peumans WJ, van Damme E & Boulter D. 1995. Expression of snowdrop lectin in transgenic tobacco plants results in added protection against aphids. Transgenic Research 4, 18-25.
Hill AV. 1937. Yellow dwarf of tobacco in Australia, I. Symptoms. Journal of Council of Scientific and Industrial Research 10 , 228-230.
Hill AV. 1941. Yellow dwarf of tobacco in Australia, II. Transmission by the jassid Thamnotettix argentata (Evans). Journal of the Council for Scientific and Industrial Research 14 , 181-186.
Hutton EM & Grylls NE. 1956. Legume 'little leaf', a virus disease of subtropical pasture species. Australian Journal of Agricultural Research 7, 85-97.
Jouanin L, Bonade-Bottino M, Girard C, Morrot G & Giband M. 1998. Transgenic plants for insect resistance. Plant Science 131 , 1-11.
Lin LC & Hou RF. 1981. Dietary requirements of Nephotettix cinciteps for amino acids and cholesterol. Chinese Journal of Entomology 1, 41-53.
150
Mitsuhashi J. 1974. Methods for rearing leafhoppers and planthoppers on artificial diets. Review of Plant Protection Research 7, 57-67.
Mitsuhashi J & Koyama K. 1972. Artificial rearing of the smaller brown planthopper, Laodelphax striatellus Fallén, with special reference to rearing conditions for the first instar nymphs. Japanese Journal of Applied Entomology and Zoology 16 , 8-17.
Moran JR & Rodoni B. 1999. Strategies for the control of tobacco yellow dwarf virus. Report on a consultancy conducted on behalf of the Tobacco Corporation of Victoria. 1-5.
Needham PD, Atkinson RG, Morris BAM, Gardner RC & Gleave AP. 1998. GUS expression patterns from a tobacco yellow dwarf virus-based episomal vector. Plant Cell Reports 17 , 631-639.
Osmelak JA. 1986. Assessment of various insecticides for the control of the vector Orosius argentatus (Evans) (Homoptera: Cicadellidae) and tomato big bud diseases. Department of Agricultural and Rural Affairs. Research Report , 1-32.
Paddick RG & French FL. 1972. Suppression of tobacco yellow dwarf with systematic organophosphorus insecticide. Australian Journal of Experimental Agriculture and Animal Husbandry 12 , 331-334.
Paddick RG, French FL & Turner PL. 1971. Control of leafhopper-borne plant disease possibly due to direct action of systemic biocides on mycoplasmas. Plant Disease Reporter 55 , 291-293.
Peferoen M. 1997. Insect control with transgenic plants expressing Bacillus thuringiensis crystal proteins. In: Carozzi, N., Koziel, M. (Eds.), Advances in insect Control: The Role of Transgenic Plants. Taylor and Francis London, 21-48.
Peumans WJ & Van Damme EJM. 1995. Lectins as plant defence proteins. Plant Physiology 109 , 347-352.
151
Pilkington LJ, Gurr GM, Fletcher MJ, Nikandrow A & Elliott E. 2004. Vector status of three leafhopper species for Australian lucerne yellows phytoplasma. Australian Journal of Entomology 42 , 366-373.
Powell KS, Gatehouse AMR, Hilder VA & Gatehouse JA. 1993. Antimetabolic effects of plant lectins and plant and fungal enzymes on the nymphal stages of two important rice pests, Nilaparvata lugens and Nephotettix cinciteps . Entomologia Experimentalis et Applicata 66 , 119-126.
Powell KS, Gatehouse AMR, Hilder VA & Gatehouse JA. 1995a. Antifeedant effects of plant lectins and an enzyme on the adult stage of the rice brown planthopper, Nilaparvata lugens . Entomologia Experimentalis et Applicata 75 , 51-59.
Powell KS, Gatehouse AMR, Hilder VA, Van Damme EJM, Peumans WJ, Boonjawat J, Horsham K & Gatehouse JA. 1995b. Different antimetabolic effects of related lectins towards nymphal stages of Nilaparvata lugens . Entomologia Experimentalis et Applicata 75 , 61-65.
Powell KS & Gatehouse JA. 1996. Mechanism of mannose-binding snowdrop lectin for use against brown planthopper in rice. In: Rice Genetics III: Proceedings of the Third International Rice Genetics Symposium (ed GS Khush) 753-758. International Rice Research Institute, Manila, Philippines.
Powell KS, Spence J, Bharathi M, Gatehouse JA & Gatehouse AMR. 1998. Immunohistochemical and developmental studies to elucidate the mechanism of action of the snowdrop lectin on the rice brown planthopper, Nilaparvata lugens (Stål). Journal of Insect Physiology 44 , 529-539.
Rao KV, Rathore KS, Hodges TK, Fu X, Stoger E, Sudhakar D, Williams S, Christou P, Bharathi M, Bown DP, Powell K, Spence J, Gatehouse AMR & Gatehouse JA. 1998. Expression of snowdrop lectin (GNA) in transgenic rice plants confers resistance to rice brown planthopper. The Plant Journal 15 , 469- 477.
152
Sauvion N, Rahbé Y, Peumans WJ, Van Damme EJM, Gatehouse JA & Gatehouse AMR. 1996. Effects of GNA and other mannose binding lectins on development and fecundity of the peach potato aphid Myzus persicae . Entomologia Experimentalis et Applicata 79 , 285-293.
Sharon N & Lis H. 1989. Lectins , Chapman & Hall, London.
Singh P. 1977. Artificial diets for insects, mites, and spiders , IFI / Plenum Data Company, New York.
Sogawa K. 1972. Studies on the feeding habits of the brown planthopper: III. Effects of amino acids and other compounds on the sucking response. Japanese Journal of Applied Entomology and Zoology 16 , 1-7.
Sogawa K. 1977. Feeding physiology of the brown planthopper. In: The rice brown planthopper pp 95-116. Food and Fertilizer Technology Center for the Asia and Pacific Region, ASPAC, Taipei, Taiwan.
Stoger E, Williams S, Christou P, Down RE & Gatehouse JA. 1999. Expression of the insecticidal lectin from snowdrop ( Galanthus nivalis agglutinin; GNA) in transgenic wheat plants: effects on predation by the grain aphid Sitobion avenae . Molecular Breeding 5, 65-73.
Thomas JE. 1979. Tobacco yellow dwarf and bean summer death viruses. PhD Thesis, University of Sydney, Australia .
Vanderzant ES. 1974. Development, significance and application of artificial diets for insects. Annual Review of Entomology 19 , 139-160.
Wang Z, Zhang K, Sun X, Tang K & Zhang J. 2005. Enhancement of resistance to aphids by introducing the snowdrop lectin gene gna into maize plants. Journal of Biosciences 30 , 627 - 638.
153
154
CHAPTER 7
ELECTROPHYSIOLOGICAL MONITORING OF THE FEEDING BEHAVIOUR OF OROSIUS ORIENTALIS ON AN ARTIFICIAL DIET AND SELECTED HOST- PLANTS
Piotr Tr ębicki 1, 2 , Rob M. Harding 1, Brendan Rodoni 3,
& Kevin S. Powell 2
1Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Queensland, GPO Box 2434, Brisbane, 4001, Australia. 2Department of Primary Industries, Biosciences Research Division, RMB 1145, Chiltern Valley Road, Rutherglen, Victoria, 3685, Australia. 3Department of Primary Industries, Private Bag 15, Ferntree Gully Delivery Centre, Knoxfield, Victoria, 3180, Australia.
155
STATEMENT OF AUTHORSHIP
Piotr Tr ębicki (principal author): Executed the work (designed and conducted all laboratory experiments, analysed and interpreted data) and wrote initial manuscript.
Signed ……………………………………. Date …18-12-09…………………..
Rob Harding: Conceived project idea, critically interpreted data and contributed to final manuscript.
Signed ……………………………………. Date …18-12-09…………………..
Brendan Rodoni: Conceived project idea, critically interpreted data and contributed to final manuscript.
Signed ……………………………………. Date …18-12-09…………………..
Kevin Powell: Conceived project idea, supervised the work, critically interpreted data and contributed to final manuscript.
Signed ……………………………………. Date …18-12-09…………………..
156
Abstract
The common brown leafhopper Orosius orientalis (Hemiptera: Cicadellidae) is a polyphagous vector of a range of economically important pathogens, including phytoplasmas and viruses, which infect a diverse range of crops. Studies on the feeding behaviour of O. orientalis were conducted using an electrical penetration graph (EPG) technique to assist in the characterisation of pathogen acquisition and transmission. Waveforms representing different feeding activities were acquired by EPG from adult O. orientalis feeding in planta, using host species Nicotiana tabacum and Phaseolus vulgaris, and in vitro using a simple sucrose- based artificial diet. Five waveforms (O1-O5) were evident when O. orientalis fed on P. vulgaris , whereas only four waveforms (O1-O4) and three waveforms (O1- O3) were observed when the leafhopper fed on N. tabacum and on the artificial diet, respectively. Both the mean duration of each waveform and waveform type differed markedly depending on the food substrate. Waveform O4 was not observed on the artificial diet and occurred relatively rarely on tobacco plants when compared with bean plants. Waveform O5 (designated as phloem ingestion) was only observed with leafhoppers feeding on bean thus providing preliminary evidence that the phloem restricted TbYDV cannot be acquired from tobacco. The attributes of the waveforms and comparative analyses with previously published Hemipteran data is presented and discussed.
157
Introduction
Many sap-sucking Hemiptera constitute economically important pests as a direct result of their feeding damage to crop plants and/or their ability to act as efficient vectors of plant pathogens. Despite their significance, there is a lack of effective control measures for many Hemiptera and/or the pathogens they vector. In many cases, this is due to a lack of knowledge of insect feeding behaviour and the mechanisms of pathogen acquisition and transmission. The development of AC (McLean & Kinsey 1964) and DC (Tjallingii 1985a, 1978, 1988) Electrical penetration graph (EPG) systems has provided a means of analysing the intra- and intercellular feeding process of many sap-sucking insects. The EPG electrophysiological monitoring approach provides real time information on insect feeding behaviour in the form of EPG waveforms, which can be correlated to specific feeding patterns and feeding locations. This method has been widely used on aphids for which there are a number of well characterised EPG waveforms which represent specific feeding activities and stylet locations in host-plant tissue (Tjallingii 1978, 1988; Tjallingii & Hogen Esch 1993). Such an approach has been used to study host-plant interactions, pathogen transmission and acquisition, insecticide mode of action, host-plant resistance and insect-induced host-plant resistance (Harrewijn & Kayser 1997; Liu et al. 2005; Prado & Tjallingii 1994a; Prado & Tjallingii 2007; van Helden & Tjallingii 2000). Although mainly used with aphids, this technique has also been adopted for other sap-sucking insect groups including planthoppers (Khan & Saxena 1988; Kimmins 1989; Powell & Gatehouse 1996; Seo et al. 2009), whiteflies (Walker & Janssen 2000), phylloxerids (Harrewijn et al. 1998; Kingston 2007), mealybugs (Calatayud et al . 2001) and thrips (Joost & Riley 2005; Kindt et al. 2006). Feeding behaviour studies on leafhoppers have been more limited but have used both the AC EPG system (Backus et al. 2005) and the DC EPG system (Kimmins & Bosque-Perez 1996; Lett et al. 2001; Miranda et al. 2009; Stafford & Walker 2009). The common brown leafhopper Orosius orientalis (Matsumura) (Hemiptera: Cicadellidae) is a highly polyphagous, sap-sucking insect able to feed on over 60 plant species (Tr ębicki et al. 2010). Apart from feeding on many
158
economically important crops both in Australia and other regions of the world, this insect is a very effective vector of numerous plant viruses and phytoplasmas. It is considered one of the most important leafhopper vectors in Australia where it transmits the phytoplasmas responsible for causing legume little leaf (Hutton & Grylls 1956), tomato big bud (Hill & Mandryk 1954; Osmelak 1986), lucerne witches broom (Helson 1951), potato purple top wilt (Grylls 1979; Harding & Teakle 1985) and Australian lucerne yellows diseases (Pilkington et al. 2004). In addition it also transmits Tobacco yellow dwarf virus (TbYDV; genus Mastrevirus , family Geminiviridae ) which causes summer death and yellow dwarf diseases in beans and tobacco, respectively (Ballantyne 1968). Despite the important pest status of O. orientalis , there is limited information available concerning its feeding behaviour, the characteristics of TbYDV acquisition and transmission and the tritrophic interactions. The control of O. orientalis currently involves the indiscriminate use of environmentally hazardous chemical insecticides. Antimetabolites are gaining popularity as a potential future control strategy since these have proven effective for other sap-sucking insect pests under controlled environment conditions (Gatehouse et al. 1992; Peumans & Van Damme 1995; Powell et al. 1993) and some exhibit antifeedant activity (Powell & Gatehouse 1996). Recently, two plant lectins were identified as having antimetabolic properties towards O. orientalis in artificial diet studies (Tr ębicki et al. 2009) but the impact of these antimetabolites on the insect’s feeding behaviour is unknown. The main objective of this study was to utilise an electrophysiological approach, the DC EPG System, to characterise and interpret waveforms produced by adult O. orientalis . A secondary objective was to compare the feeding behaviour of adult O. orientalis on a simple artificial diet, a preferred host-plant (Phaseolus vulgaris L.) and non-preferred host-plant (Nicotiana tabacum L.) species. Studies using the artificial diet substrate should assist in the future assessment of potential antimetabolites on the insect’s feeding behaviour, while studies of the feeding behaviour of O. orientalis on both preferred and non- preferred host-plants will improve fundamental knowledge on the epidemiology of TbYDV.
159
Materials and methods
Insect and plant material All adult O. orientalis used in this study were obtained from stock colonies reared at Charles Darwin University, Darwin, Australia. Stock leafhopper cultures were maintained on celery (Apium graveolens L.) for several generations, and early instar nymphs were placed in rearing cages (Tr ębicki et al. 2009) until adulthood. Tobacco and beans were maintained in an insect-free, climate-controlled environment (25 + 3°C) and used at the 2-4 leaf stage for all plant EPG recordings.
Data collection The feeding behaviour of O. orientalis was monitored by the EPG system (Tjallingii 1988) using a Giga-8 DC amplifier (direct current) (EPG-Systems, Wageningen, The Netherlands) with a 1 Giga Ώ input resistance. All recordings were conducted within a Faraday cage housed within a climate-controlled laboratory room (22 + 3°C). EPG output was set to 100x gain, data was initially acquired at 122 Hz (later converted to 100 Hz) using a Di700 A/D data acquisition card (Dataq® instruments, Ohio, USA). Data was analysed using Probe 3.4 for Windows software (Department of Entomology, Wageningen University, The Netherlands). As O. orientalis were active when disturbed, each insect was immobilized prior to electrode attachment by chilling at -20°C for 90 s in a 5 ml screw top plastic tube. To ensure that cold treatment did not affect insect behaviour, pilot experiments were conducted at different temperatures and durations and no negative effects were observed (data not presented). Immobilized insects were then transferred onto a vacuum device (van Helden and Tjallingii 2000) platform for tethering. Insects were tethered to the electrode using a small droplet of water -based silver glue (EPG-Systems, Wageningen, The Netherlands) placed on the pronotum using a fine entomological pin. After 20 s, a second droplet of silver glue was added and a gold wire (12.5 µm diameter, 3 cm length) was placed in the glue and allowed to dry, the unsecured end of the gold wire was attached by silver glue to a brass pin which was connected to the EPG insect
160
electrode. Each wired leafhopper was left tethered for 1 h and then placed in the centre of the feeding substrate (either bean, tobacco or an artificial diet). Depending on insect availability, 20-22 samples per treatment were evaluated sequentially in a randomised pattern and each EPG recording covered an 8 h time interval. All recordings were done using the same setting, with plant voltage adjusted for each channel ensuring the first insect probe was always positive with maximum amplitude of around +4 V. Electrical origin for most of the waveforms was determined by changing the targeted waveform voltage above and below 0 V level. In addition, to determine the electrical origin, a simultaneous recording of O. orientalis feeding was obtained using a combined AC/DC EPG amplifier (Wageningen University, The Netherlands) and waveforms were directly compared at the same time point and identified as either resistance (R) or electromotive force (emf) in origin. For each recording, the quality of silver glue connection between leafhopper and insect electrode was tested by using a calibration pulse after the first probe was initiated and a good contact was determined by an output signal in the form of a square pulse.
Artificial diet and diet chamber An artificial diet comprising a 5% (w/v) sucrose solution was used to study the feeding behaviour of O. orientalis. Sucrose was dissolved in sterile ultra-pure water using gentle heat (25°C) on a magnetic stirrer hotplate, and the pH adjusted to 6.5 with 1M KOH. After filtration through a 0.2 µm Millipore disposable filter, diet solutions were dispensed into 250 ml plastic containers as stock solutions and further dispensed into 50 ml tubes as working solutions and stored at -20°C prior to use. An artificial diet feeding platform was constructed from a small plastic Petri dish (1 cm x 3.5 cm) with an access hole in the side to allow contact with the EPG diet electrode. The Petri dish was filled to capacity with the sucrose diet and a single layer of Parafilm M™ was stretched over the chamber carefully to prevent the occurrence of air bubbles (Figure 7.1). A single adult O. orientalis was attached to the EPG electrode as previously described and placed on the top of the Parafilm M™ layer in the centre of the feeding platform. Orosius orientalis stylet movement inside and outside of the feeding platform was observed using a light microscope
161
Figure 7.1 Chamber to study the feeding behaviour of Orosius orientalis on an artificial diet. One electrode is connected to the leafhopper while the other is placed in the diet through a side-opening which is sealed post electrode insertion to prevent diet leakage.
162
Data analysis Statistical analyses were done on each individual insect EPG recording as well as on combined recordings for each treatment. Online resources to calculate EPG parameters were used (Giordanengo 2009) as well as summary statistics using Genstat software (10 th Edn 2007, Lawes Agricultural Trust, Rothampsted, UK).
Results
Using EPG, the feeding behaviour of O. orientalis was studied on a preferred and non-preferred host-plant (bean and tobacco, respectively) and on a simple artificial diet. Over each 8 h experimental period, five distinctive waveform patterns were identified which were designated O1-O5 (Figure 7.2) in accordance with the order they generally appeared during the feeding process. In addition, a non-probing phase (np) was observed together with potential drops (pd). EPG waveforms were distinguished based on voltage level, frequency, amplitude and shape (Table 7.1).
EPG waveforms on bean, a preferred host-plant After an initial non-probing phase (np), stylet penetration always commenced with waveform O1 (Figure 7.3). Depending on the plant voltage adjustment, this waveform was characterised by sharp positive peaks, very often accompanied by a relatively large number of spikes with a gradual decline in voltage level. Amplitude, voltage and shape was highly variable over the experimental period but the waveform remained positive (above 0 V) for its entire duration (Table 7.1).
163
Figure 7.2 Visual representation of the distinctive electrical penetration graph waveforms produced by adult Orosius orientalis feeding on bean (O1-O5), tobacco (O1-O4) and an artificial diet (O1-O3); pd = potential drop.
164
Table 7.1 Major characteristics of the waveforms recorded using a DC EPG system for adult Orosius orientalis feeding on beans (waveforms O1-O5), tobacco (O1-O4) or an artificial diet (O1-O3).
Waveform characteristics Proposed correlation* Relative Repetition EPG Voltage Electrical amplitude rate Plant tissue Remarks waveform level a origin b (%) (Hz)
Epidermis, parenchyma, all Cuticle penetration, sheath salivation, O1 100 Variable E R tissues pathway activity Cell puncture by stylet, possibly pd - na I emf All living cells salivation and ingestion Active feeding probably from xylem or O2 35 4-5 E R/emf Xylem and/or parenchyma parenchyma
O3 50 Variable E R Undetermined Unknown
O4 - 0.6-1 I emf Sieve elements Unknown – possibly watery salivation
O5 - 2 I unknown Sieve elements Unknown - possibly phloem ingestion
a E =extracellular (positive), I = intracellular (negative); b R = resistance, emf = electromotive force pd = potential drop; na = not applicable; * based on comparison with published studies on other Hemipteran insects.
165
Waveform O2 always occurred after O1 and less frequently than O1 (Figure 7.3). The waveform remained positive (Table 7.1) with an amplitude ranging from 1-4 V. It was characterised by a monophasic waveform with regular periodic signals and a smooth peak and sharp basal troughs. The relatively high amplitude at the beginning of the waveform remained at a constant level for durations of up to 30 minutes, but for longer time periods, the amplitude gradually decreased. Waveform O3 was usually preceded by waveform O2 and occurred later in the feeding process (Figure 7.3). The voltage level of this waveform was always positive (Table 7.1). This waveform was characterised by a smooth, flat line with irregular, occasional interruptions of rapid, sharp peaks with amplitude significantly higher than the line itself. From the peak, the waveform gradually increased in voltage level. This waveform occurred only occasionally and less frequently than waveform O2. A relatively sustained (ie longer that 10 minutes) waveform O4 appeared much later into the feeding process with initial short probes present from 3500 seconds (Figure 7.3). This waveform always occurred after O1 and O2. O4 occurred less frequently than other waveforms but, although absent from a few recordings, its duration sometimes exceeded one hour. The O4 waveform occurred following rapid sharp negative voltage (<0 V) and generally remained negative with the exception of a small number of recordings when it had a tendency for a gradual movement towards 0 V without ever exceeding it. This represented a relatively complex waveform characterised by a “horizontal line” proceeded by upward peaks and downward troughs. The frequency of the whole waveform was low (<1 Hz) with a high frequency around 14 Hz of the “horizontal line” between upward and downward peaks (Table 7.1). Waveform O5 was the least frequent of all waveforms and was mainly preceded by waveform O4. In a few recordings, this was interrupted by short periods (less than 3 min) of waveform O2. The voltage level of waveform O5 was always negative and relatively constant, with relatively small amplitude of around 250 mV and a 2 Hz frequency. This waveform exhibited sharp negative peaks that were mostly regular although they varied in amplitude (Figure 7.1, Table 7.1). O1 was not only the most frequently observed waveform but also had the shortest mean duration of all waveforms. In contrast, waveforms O4 and O5 had
166
the longest mean duration but were only observed infrequently; waveform O5, for example, was only recorded on average once per insect/plant combination (Figure 7.4, 7.5).
EPG waveforms on tobacco, a non-preferred host-plant With O. orientalis feeding on tobacco plants, only four distinctive feeding activities were identified, represented by the previously described waveforms O1- O4, as well as potential drops and non-probing (np) phases. Excluding the np phases, the duration of waveform O3 was the longest followed by waveforms O2 and O1 (Figure 7.5). Waveform O1 occurred most frequently (an average 25 times per recording), while O4 was the both the shortest (mean duration of 13 s) and least frequently (mean number of probes = 0.3) observed waveform (Figure 7.4, 7.5). Waveform O5 was not observed in any EPG recording.
167
Figure 7.3 Mean time taken for Orosius orientalis to reach waveforms O1-O5 when placed on an artificial diet, tobacco plants and bean plants.
168
Figure 7.4 Mean number of feeding waveforms (O1-O5) and non-probing (np) phases occurring from Orosius orientalis feeding on an artificial diet, tobacco plants and bean plants.
169
Figure 7.5 Mean duration of waveforms (O1-O5) and non-probing (np) phases occurring from Orosius orientalis feeding on an artificial diet, tobacco plants and bean plants.
170
EPG waveforms on an artificial diet The feeding behaviour studies of O. orientalis in vitro were conducted using a simple 5% sucrose artificial diet under negative pressure. Three distinctive waveform patterns (the previously described O1-O3) and np periods were identified in all recordings but potential drops were absent. Of all the waveforms observed, O2 occurred for the longest period with a mean duration of 2517 seconds followed by waveforms O3 and O1, respectively (Figure 7.5). During waveform O1, a clear watery secretion was observed on the Parafilm membrane of the artificial diet chamber soon after contact with the insects’ labium.
Comparison of feeding behaviour on an artificial diet and host-plants The first phase in EPG recordings is usually a non-probing phase (np) which occurs prior to the insects stylet being inserted into the feeding substrate. The np phase was observed in all recordings from bean, tobacco and the artificial diet. However, there were quite marked differences in waveform events and duration depending on the feeding substrates. The mean number of np events was three fold lower on the artificial diet than on bean and tobacco plants (Figure 7.4). In contrast, the mean duration of np on the artificial diet was markedly longer, almost double that recorded on both tobacco and beans (Figure 7.5). The shortest mean duration of np was recorded on the preferred host bean. The duration of waveforms O1-O3, particularly O2 and O3, was much longer with insect’s feeding on the sucrose diet compared to plants (Figure 7.5). In terms of waveform frequency, waveform O1 occurred less frequently on the artificial diet than on tobacco and bean (Figure 7.4). On bean, this waveform was the most frequent (Figure 7.4) and had the shortest mean time till first recorded and mean duration in comparison to the sucrose diet and tobacco. On beans, it was recorded twice as often as on the artificial diet and the total time spent as O1 was almost double when feeding on plants (bean and tobacco) compared to the artificial diet (Figure 7.3, 7.5). Waveform O2 events were recorded more frequently (up to three-fold) on the artificial diet than on either bean or tobacco (Figure 7.4). In addition, the mean duration of O2 was almost five times longer on the artificial diet than on beans and 10 times longer than on tobacco plants (Figure 7.5). Additionally, O.
171
orientalis took longer to reach this waveform on tobacco than on diet and bean plant (Figure 7.3) The duration of waveform O3 was over two and three times longer on the artificial diet than on tobacco and bean, respectively (Figure 7.5). The mean number of O3 events was markedly higher on the artificial diet than on tobacco and beans (Figure 7.4), although the time to reach this waveform was longer on the artificial diet (Figure 7.3). Waveform O4 was never observed in any recordings using the artificial diet and was only recorded on beans and tobacco. On tobacco, the mean number and duration was very low and significantly different to bean (0.3 times and 13 s, respectively). Despite a relatively low number of O4 events on bean, the mean duration was relatively long compared to tobacco (Figures 7.4, 7.5). Orosius orientalis took less time to reach this waveform on beans than on tobacco (Figure 7.3). Waveform O5 was only recorded from O. orientalis feeding on beans. On average, at least one O5 event was recorded per bean EPG recording and this event was relatively long, being in excess of 608 s. This waveform usually occurred relatively late in the feeding process (Figures 7.3, 7.4, 7.5).
Discussion
This is the first study to investigate the feeding behaviour of O. orientalis using an EPG approach. Studies on the feeding behaviour characteristics of this leafhopper using the DC EPG system were conducted on bean, a preferred host-plant (in which TbYDV can be both acquired and transmitted), tobacco, a non-preferred host-plant (in which TbYDV can be transmitted but not acquired) and a simple sucrose-based artificial diet. A total of five distinctive waveforms, designated O1- O5, were observed depending on the feeding substrate, along with potential drops and non-probing phases. The characteristics of the five waveforms showed some distinct similarities to those observed for other leafhoppers, planthoppers and aphids (Kimmins & Bosque-Perez 1996; Lett et al . 2001; Stafford & Walker 2009; Tjallingii 1978).
172
Due to a large variation in waveform characteristics between leafhopper species, there is no universally accepted nomenclature for leafhopper EPG waveforms although a standard nomenclature has been assigned for aphids (Tjallingii 1978). Feeding waveforms for the leafhopper, Cicadulina storey, have been named L1-L5 (Kimmins & Bosque-Perez 1996) while those from C. mbila are designated as 1-5 (Lett et al . 2001). For the leafhopper, Circulifer tenellus, a more complicated waveform nomenclature has been reported which includes a pathway phase (waveforms A, B1, B2 and C), a non-phloem ingestion phase (waveform G) and a phloem ingestion phase (waveforms D1, D2, D3 and D4) (Stafford and Walker 2009). In this study, we have tentatively proposed the terminology O1-O5 to describe the feeding waveforms which is based on the first letter of the genus name (O = Orosius ) with each number representing a different waveform type which is likely to be related to a different feeding activity. Waveform O1 always occurred after a non-probing phase and was recorded from insect’s feeding on all three substrates. Similar waveforms have been observed with aphids (Tjallingii 1978) and other leafhopper species (Kimmins & Bosque-Perez 1996; Lett, et al . 2001; Stafford & Walker 2009) and are reported to represent the pathway phase. During this phase, the insect produces gelling saliva that creates the salivary sheath and lubricates the stylet as it advances towards the vascular bundle. Interestingly, O. orientalis -derived secretions were clearly observed on the Parafilm membrane of the artificial diet layer soon after contact of the insects’ labium with the membrane during this waveform. This supports the notion that waveform O1 most likely corresponds to the pathway phase. On both plant species, but not on the artificial diet, waveform O1 was frequently interrupted by potential drops. Drops in the voltage potential are known to occur during intracellular punctures in the pathway phase before the phloem is reached (Tjallingii 1985b; Tjallingii & Gabry ś 1999) and, as such, would explain their absence from the artificial diet EPG recordings. The membrane potential in all living plant cells, including phloem cells, is positive outside and negative inside. Puncture of the membrane by the stylet tip and subsequent stylet movement inside the plant cell results in rapid changes of voltage during EPG recordings (Tjallingii 1985b). From aphid recordings, potential drops are characterised by a sudden drop of voltage of around 100 mV after puncture of the cell and a steep rise to extracellular voltage after stylet
173
withdrawal from the cell. Sudden drops in voltage potential were observed in all O. orientalis recordings on bean and tobacco but, in contrast with aphid recordings, this was followed by a slow and gradual rise at the extracellular level. It is possible that damage to the cell membrane by the relatively large stylet of O. orientalis is much greater than with aphids and this may have caused the collapse and gradual leakage of cell contents and slow movement of voltages towards a positive level. Waveform O2 was observed in all artificial diet and plant EPG recordings. It was very similar to waveform G observed in aphids (Prado & Tjallingii 1994a; Tjallingii 1988) and whiteflies (Lei et al. 1999), waveform N5 reported in the rice brown planthopper, Nilavarvata lugens (Seo et al . 2009) and waveforms 1, G and Xc from the leafhoppers C. mbila , C. tenellus and Bucephalogonia xanthophis, respectively (Lett et al . 2001; Miranda et al . 2009; Stafford & Walker 2009). In all cases, this waveform have been correlated with active feeding from xylem and/or the mesophyll. The presence of waveform O2 from O. orientalis feeding on the artificial diet, which has a negative hydrostatic pressure, as well as from both plants, indicates that O. orientalis may be actively ingesting fluid and that this pattern may be associated with the rhythmic activity of cibarial muscle when ingesting fluid, in the form of either diet or sap. The fact that no changes in voltage level were recorded, which typically indicates the stylet puncturing a living cell, suggests that this waveform is most likely to be a xylem-related activity in planta . This waveform was also the most prevalent form recorded from leafhoppers feeding on the artificial diet. This may be due to the presence of sucrose which is a known sucking stimuli for other sap-sucking insects including, N. lugens (Sogawa 1982). Waveform O3 was recorded from leafhoppers feeding on all three substrates but was most commonly seen using the artificial diet. A similar waveform has been reported for other leafhoppers and planthoppers, including C. mbila (Lett et al . 2001), but both the feeding activity and stylet tip location associated with the waveform remains unknown. Waveform O4 was only recorded from leafhoppers feeding on plants, although the occurrence and duration of this waveform on tobacco was relatively low (Figure 7.4, 7.5). This waveform was always preceded by a drop in voltage, similar to a potential drop, and remained below 0 V for its entire duration. Similar
174
waveforms have been recorded for the leafhoppers, C. mbila and C. storeyi (Kimmins & Bosque-Perez 1996; Lett et al . 2001), with waveform L2 of C. storeyi associated with the transmission of the phloem-restricted geminivirus, Maize streak virus. As such, this waveform may be associated with the salivary pump action of injecting saliva-containing virus particles into phloem cells (Kimmins & Bosque-Perez 1996). Another similar waveform to O4 is waveform E1 reported in aphids. This waveform has been correlated with phloem activity and watery saliva secretion associated with Barley yellow dwarf virus transmission (Prado & Tjallingii 1994a). The absence of waveform O4 from O. orientalis feeding on the artificial diet under negative pressure provides circumstantial evidence that this waveform may be related to phloem activity as the necessity to salivate is triggered by the relatively high pressure present in phloem tissue. Although the feeding behaviour of O. orientalis , and many other leafhopper species, is poorly understood it is possible that, for many leafhopper species, a number of phloem-related plant properties and reactions need to be overcome to enable feeding on phloem sap. These include coagulating proteins which are up regulated as a host-plant wound response. The role of watery saliva secretions into the phloem prior to sap uptake by aphids was suggested as a necessary step to suppress the wound response and cascade of reactions caused by insect-induced cell disruption (Tjallingii 2006; Will et al. 2007). Waveform O5 was only present in recordings from O. orientalis feeding the preferred host-plant, bean, and although it was relatively less common than other waveforms it was relatively longer in duration. This waveform occurred after O4 with a similar median voltage to O4 which was below 0 V. Waveform O5 resembles phloem ingestion waveforms reported for C. mbila and is very similar to the E2 phloem waveform observed with aphids (Lett et al . 2001). Many phloem feeders, especially aphids, take advantage of the high hydrostatic pressure in the phloem to passively ingest the sap (Lett et al . 2001; Prado & Tjallingii 1994b). To achieve this, the large cybarial muscles that are used during active feeding (i.e. during xylem phase) are substituted by a precybarial valve that, by opening and closing, regulates the amount of phloem entering the food canal (McLean and Kinsey 1984). The fact that the O5 waveform was not recorded on the artificial diet is presumably due to the lack of sufficient hydrostatic pressure that forces the insect to actively feed.
175
O. orientalis is a successful vector of many viruses and phytoplasmas including the phloem-restricted TbYDV. Although O. orientalis can acquire the virus from bean and other host-plants, it cannot acquire the virus from TbYDV- infected tobacco plants (Helson 1950). Since the virus particles are introduced into plant sap with saliva during the salivation phase, and are acquired during ingestion, the presence of waveform O4 and absence of waveform O5 from leafhoppers feeding on tobacco provides further circumstantial evidence that O4 is associated with phloem location of the stylet and salivation, while O5 represents an ingestion phase. The recent development of an artificial diet for O. orientalis (Tr ębicki et al. 2009; Chapter 6) will enable further studies to be undertaken under negative and positive hydrostatic pressure to further understand the mechanism of action of antimetabolites. Additional research also needs to be conducted to further characterise the waveforms and correlate them to specific feeding activities on both virus-infected and non-infected host plants. Such studies would require a histological approach for the plant and insect combined with laser stylectomy and time lapse videophotography of the insect. An understanding of the physiological meaning of the waveforms will be critical to understanding the feeding behaviour and the mechanisms of pathogen transmission/acquisition of this very important vector.
Acknowledgements
This research was funded by Horticulture Australia Limited and the Tobacco Research and Development Corporation with in-kind support from the Department of Primary Industries (DPI), Victoria, Australia. The assistance of Prof. Freddy Tjallingii (EPG-Systems, Wageningen, The Netherlands), Dr Kim Andrews (DPI Rutherglen), Gary Baxter (DPI Ovens) and Lucy Tran-Nguyen (Charles Darwin University, Australia) is gratefully acknowledged.
176
References
Backus EA, Habibi J, Yan F & Ellersieck M. 2005. Stylet penetration by adult Homalodisca coagulata on grape: electrical penetration graph waveform characterization, tissue correlation, and possible implications for transmission of Xylella fastidiosa . Annals of the Entomological Society of America 98 , 787-813.
Ballantyne B. 1968. Summer death of beans. Agricultural Gazette of New South Wales 79 , 486-489.
Calatayud P-A, Seligmann CD, Polania MA & Bellotti AC. 2001. Influence of parasitism by encyrtid parasitoids on the feeding behaviour of the cassava mealybug Phenacoccus herreni . Entomologia Experimentalis et Applicata 98 , 271-278.
Day MF, Irzykiewicz H & McKinnon A. 1952. Observations on the feeding of the virus vector Orosius argentatus (Evans), and comparisons with certain other jassids. Australian Journal of Scientific Research , Series B - Biological Sciences 5 , 128-142.
Day MF & McKinnon A. 1951. A study of some aspects of the feeding of the jassid Orosius . Australian Journal of Scientific Research , Series B - Biological Sciences 4 , 125-135.
Gatehouse AMR, Boulter D & Hilder VA. 1992. Potential of plant-derived genes in the genetic manipulation of crops for insect resistance. In: Plant Genetic Manipulation for Crop Protection. (Eds AMR Gatehouse, D Boulter & VA Hilder), CAB International, Wallingford, United Kingdom, 135-153.
Giordanengo P, 2009. EPG-Calc 4.8, a php program to calculate EPG parameters. Université de Picardie Jules Verne, Amiens, France. http://www.u- picardie.fr/PCP/UTIL/epg.php .
177
Grylls NE. 1979. Leafhopper vectors and the plant disease agents they transmit in Australia. In: Leafhopper Vectors and Plant Disease Agents, (Eds K Maramorosch & KF Harris), Academic Press, New York, 179–214.
Harding RM & Teakle DS. 1985. Mycoplasma-like organisms as causal agents of potato purple top wilt in Queensland. Australian Journal of Agricultural Research 36 , 443-449.
Harrewijn P & Kayser H. 1997. Pymetrozine, a fast-acting and selective inhibitor of aphid feeding. In-situ studies with electronic monitoring of feeding behaviour. Pesticide Scienc e 49 , 130-140.
Harrewijn P, Piron PGM & Ponsen MB. 1998. Evolution of vascular feeding in aphids: an electrophysiological study. P roceedings of Experimental and Applied Entomology 9, 29-34.
Helson GAH. 1951. The transmission of witches broom virus disease of lucerne by the common brown leafhopper, Orosius argentatus (Evans). Australian Journal of Scientific Research , Series B - Biological Science 4, 115-124.
Helson GAH. 1950. Yellow dwarf of tobacco in Australia V. Transmission by Orosius argentatus (Evans) to some alternative host-plant. Australian Journal of Agricultural Research 1, 144-147.
Hill AV & Mandryk M. 1954. A study of virus diseases "big bud" of tomato and "yellow dwarf" of tobacco. Australian Journal of Agricultural Research 5, 617- 625.
Hutton EM & Grylls NE. 1956. Legume 'little leaf', a virus disease of subtropical pasture species. Australian Journal of Agricultural Research 7, 85-97.
Joost PH & Riley DG. 2005. Imidacloprid effects on probing and settling behavior of Frankliniella fusca and Frankliniella occidentalis (Thysanoptera: Thripidae) in tomato. Entomologia Experimentalis et Applicata 98 , 1622-1629.
178
Khan ZR & Saxena RC. 1988. Probing behaviour of three biotypes of Nilaparvata lugens (Homoptera: Delphacidae) on different resistant and susceptible rice varieties. Journal of Economic Entomology 81 , 1338-1345.
Kimmins FM. 1989. Electrical penetration graphs from Nilaparvata lugens on resistant and susceptible rice varieties. Entomologia Experimentalis et Applicata 50 , 69-79.
Kimmins FM & Bosque-Perez NA. 1996. Electrical penetration graphs from Cicadulina spp. and the inoculation of a persistent virus into maize. Entomologia Experimentalis et Applicata 80 , 46-49.
Kindt F, Joosten NN & Tjallingii WF. 2006. Electrical penetration graphs of thrips revised: Combining DC- and AC-EPG signals. Journal of Insect Physiology 52 , 1-10.
Kingston KB. 2007. Digestive and feeding physiology of grape phylloxera (Daktulosphaira vitifoliae Fitch). PhD thesis, Faculty of Science, Australian National University, Canberra, Australia. 281p.
Lei H, van Lenteren JC & Tjallingii WF. 1999. Analysis of resistance in tomato and sweet pepper against the greenhouse whitefly using electrically monitored and visually observed probing and feeding behaviour. Entomologia Experimentalis et Applicata 92 , 299–309.
Lett J-M, Granier M, Grondin M, Turpin P, Molinaro F, Chiroleu F, Peterschmitt M & Reynaud B. 2001. Electrical penetration graphs from Cicadulina mbila on maize, the fine structure of its stylet pathways and consequences for virus transmission efficiency. Entomologia Experimentalis et Applicata 101 , 93-109.
Liu XD, Zhai BP, Zhang XX & Zong JM. 2005. Impact of transgenic cotton plants on a non-target pest, Aphis gossypii Glover. Ecological Entomology 30 , 307–315.
McLean DL & Kinsey MG. 1984. The precibarial valve and its role in the feeding behavior of the pea aphid, Acyrthosiphon pisum . Bulletin of Entomological Society of America 30 , 26-31.
179
McLean DL & Kinsey MG. 1964. A technique for electronically recording aphids feeding and salivation. Nature (London) 202 1358-1359.
Miranda MP, Fereres A, Appezzato-da-Gloria B & Lopes JRS. 2009. Characterization of electrical penetration graphs of Bucephalogonia xanthophis , a vector of Xylella fastidiosa in citrus. Entomologia Experimentalis et Applicata 130 , 35-46.
Osmelak JA. 1986. Assessment of various insecticides for the control of the vector Orosius argentatus (Evans) (Homoptera: Cicadellidae) and tomato big bud diseases. Department of Agricultural and Rural Affairs. Research Report, 1-32.
Peumans WJ & van Damme EJM. 1995. Lectins as plant defence proteins. Plant Physiology 109 , 347-352.
Pilkington LJ, Gurr GM, Fletcher MJ, Nikandrow A & Elliott E. 2004. Vector status of three leafhopper species for Australian lucerne yellows phytoplasma. Australian Journal of Entomology 42 , 366-373.
Powell KS, Gatehouse AMR, Hilder VA & Gatehouse JA. 1993. Antimetabolic effects of plant lectins and plant and fungal enzymes on the nymphal stages of two important rice pests, Nilaparvata lugens and Nephotettix cinciteps . Entomologia Experimentalis et Applicata 66 , 119-126.
Powell KS & Gatehouse JA. 1996. Mechanism of action of mannose-binding snowdrop lectin for use against brown planthopper in rice. In: Rice Genetics III. Proceedings of the Third International Rice Genetics Symposium . Ed. by G S Kush, IRRI, Manila (Philippines), 753-758.
Prado E & Tjallingii WF. 1994. Aphid activities during sieve element punctures. Entomologia Experimentalis et Applicata 72 , 157-165.
Prado E & Tjallingii WF. 2007. Behavioral evidence for local reduction of aphid- induced resistance. Journal of Insect Scienc e 7, 1-8.
180
Seo BY, Kwon Y-H, Jung JK & Kim G-H. 2009. Electrical penetration graphic waveforms in relation to the actual positions of the stylet tips of Nilaparvata lugens in rice tissue. Journal of Asia-Pacific Entomology 12 , 89-95.
Sogawa K. 1982. The rice brown planthopper: feeding physiology and host-plant interactions. Annual Review of Entomology 27 , 49-73.
Stafford CA & Walker GP. 2009. Characterization and correlation of DC electrical penetration graph waveforms with feeding behavior of beet leafhopper, Circulifer tenellus . Entomologia Experimentalis et Applicata 130 , 113–129.
Tjallingii WF. 1978. Electronic recording of penetration behaviour by aphids. Entomologia Experimentalis et Applicata 24 , 721-730.
Tjallingii WF. 1985a. Electrical nature of recorded signals during stylet penetration by aphids. Entomologia Experimentalis et Applicata 38 , 177-186.
Tjallingii WF. 1985b. Membrane potentials as an indication for plant cell penetration by aphid stylets. Entomologia Experimentalis et Applicata 38 , 187- 193.
Tjallingii WF. 1988. Electrical recording of stylet penetration activities. pp. 95- 108. In: Aphids: their biology, natural enemies and control. (Eds AK Minks & P Harrewijn), Elsevier, The Netherlands,
Tjallingii WF. 2006. Salivary secretions by aphids interacting with proteins of phloem wound responses. Journal of Experimental Botany 57 , 739-745.
Tjallingii WF & Gabry ś B 1999. Anomalous stylet punctures of phloem sieve elements by aphids. Entomologia Experimentalis et Applicata 91 , 97-103.
Tjallingii WF & Hogen Esch TH. 1993. Fine structure of aphid stylet routes in plant tissue in correlation with EPG signals. Physiological Entomology 18 , 317- 328.
Tr ębicki P, Harding RM & Powell KS 2009. Antimetabolic effects of Galanthus nivalis agglutinin and wheat germ agglutinin on nymphal stages of the common
181
brown leafhopper using a novel artificial diet system. Entomologia Experimentalis et Applicata 131 , 99-105.
Tr ębicki P, Harding RM, Rodoni B, Baxter G & Powell KS 2010. Vectors and alternative hosts of Tobacco yellow dwarf virus in south-eastern Australia. Annals of Applied Biology 157 , 13-24. van Helden M & Tjallingii WF. 2000. Experimental design and analysis in EPG experiments with emphasis on plant resistant research, pp. 144-171. In: Principles and Applications of Electronic Monitoring and Other Techniques in the Study of Homopteran Feeding Behaviour. (Eds GP Walker & EA Backus), Thomas Say Publications in Entomology, USA.
Walker GP & Janssen JAM. 2000. Electronic recording of Whitefly (Homoptera: Aleyrodidae) feeding and oviposition behaviour, pp. 172-200. In: Principles and Applications of Electronic Monitoring and Other Techniques in the Study of Homopteran Feeding Behaviour . (Eds GP Walker & EA Backus), Thomas Say Publications in Entomology, USA.
Will T, Tjallingii WF, Thönnessen A & van Bel AJE. 2008. Molecular sabotage of plant defense by aphid saliva. Proceedings of the National Academy of Sciences of the United States of America 104 , 10536–10541.
182
CHAPTER 8
GENERAL DISCUSSION AND CONCLUSIONS
This study was conducted to investigate the epidemiology of tobacco yellow dwarf disease by examining the fundamental interactions between Tobacco yellow dwarf virus (TbYDV), its vector(s) and host-plants. During the course of this project, host-plants from in and around crop borders were examined both visually and using molecular techniques for the presence of TbYDV. In addition, extensive insect surveys were conducted to confirm that Orosius orientalis was the major vector and to identify other potential vectors of TbYDV. The incidence of TbYDV was monitored in both plants and insects in several field sites with different disease pressure levels. Site-specific characteristics including climatic conditions, adjacent vegetation and leafhopper diversity and relative abundance were found to influence the incidence and severity of the disease. The feeding physiology of O. orientalis was also investigated in vitro and in planta using a novel electrophysiological technique and novel rearing methods. This resulted in the development of the first published artificial diet for O. orientalis and the first characterisation of electrical waveforms associated with the insect’s feeding behaviour. This combination of developing an artificial rearing medium for O. orientalis and characterising the insect’s feeding behaviour provided the fundamental basis to an improved understanding of insect-host-plant interactions and ultimately the impact this can have on virus transmission and acquisition processes. The results from the field studies showed that the occurrence of tobacco yellow dwarf disease was influenced by a number of factors, both biotic and abiotic. The biotic factors included vector abundance and the presence of weeds, specifically Amaranthus retroflexus and Raphanus raphanistrum which can act as virus reservoirs, while the abiotic factors included rainfall and temperature. The current control strategy for TbYDV involves the regular spraying of chemical insecticide which is expensive, ineffective and harmful to the environment
183
(Osmelak 1986; Paddick & French 1972; Paddick et al. 1971). As such, knowledge regarding the timing of insect population peaks and virus presence is crucial in improving disease management. The research from this study suggests that more effective disease control might be achieved by limiting insecticide applications to summer months since this was when viruliferous O. orientalis was present in highest numbers. Additionally, disease occurrence in beans and tobacco could be reduced by eliminating the TbYDV weed hosts, Amaranthus retroflexus and Raphanus raphanistrum, which were commonly found in and around crops at the two most disease-affected sites, either by targeted herbicide application or roguing. The effectiveness of such approaches would require additional field studies. Insect vectors are the most important link in the spread of TbYDV and occurrence of tobacco yellow dwarf disease in commercial crops. Maintaining the vegetation adjacent to the crop and keeping it free of virus source plants can aid in disease management and reduce disease severity but not necessary reduce insect numbers. Many vector species, including O. orientalis , are very mobile and can migrate long distances (Helson 1942; Hosking & Danthanarayana 1988). Knowledge of population dynamics is very important in implementing efficient control options but for many leafhopper vectors, including O. orientalis, there is limited published population data. Additionally, the most suitable and effective method for monitoring leafhoppers in agricultural areas is poorly understood and the choice of sampling technique can differ between species. Based on this study, a number of different trapping methods are recommended in order to accurately monitor the population dynamics and abundance of a broad range of leafhopper species. Although yellow pan traps were found to be the most effective at catching O. orientalis , sufficient numbers of O. orientalis were still recorded in the yellow sticky trap and sweep nets to demonstrate the trimodal peak in seasonal activity that was observed at all four field sites studied. The use of pan traps would be less suitable for farmers implementing IPM due to the time required for their maintenance and processing. In this situation, sticky trap and sweep net are more suitable for accurately recording peaks of activity. The results from this research also highlight the lack of detailed data on leafhopper distribution and diversity in Australia. This was clearly evidenced by a large proportion of species collected
184
which had not been previously recorded in Victoria together with an additional 15 unidentified species which may prove to be new species. There are a number of different feeding strategies used by sap-sucking insect vectors which influence their ability to acquire and transmit pathogens between host-plants (Backus et al. 2005). Understanding the feeding requirements and behaviour of pest species, especially insect vectors, is a necessary fundamental step towards their efficient control. In many cases, insects are not considered pests if they are non-viruliferous since their impact on plants, from direct feeding alone, is minimal. Orosius orientalis is a highly polyphagous, phloem-feeding leafhopper which is responsible for vectoring many pathogens (Grylls 1979; Harding & Teakle 1985; Helson 1951; Hill 1941; 1950; Pilkington et al. 2004; Thomas & Bowyer 1980; van Rijswijk et al. 2002). Although it can feed on a broad host-plant range not all hosts allow the insect to fully develop and, at best, should be considered as only transient hosts with tobacco being a prime example (Helson 1950). Orosius orientalis can feed on tobacco long enough to transmit virions it is carrying but it cannot acquire virions from tobacco or survive on this host for an extended period. Since TbYDV is phloem-restricted, an inability to uptake the sap containing virus particles by O. orientalis from tobacco plant might indicate the presence of a phloem-based resistance factor in tobacco towards the insect. Prior to commencement of this study, there was no published data or understanding of the basic nutritional requirements of O. orientalis or the mechanism of its feeding behaviour (and therefore the transmission and acquisition characteristics of TbYDV). For this reason, one focus of this PhD project was to understand the insect's feeding requirements by developing an artificial diet and, once determined, identify possible novel control agents using an in vitro feeding system. Additionally, feeding behaviour was studied using an electrophysiological technique on host and non-host-plants and an artificial diet. This study described the first feeding system and chemically defined novel artificial diet for O. orientalis, a diet that allowed insect survival for up to 46 days from first instar through to adulthood. This artificial diet allowed the screening of three selected plant-derived proteins for their potential anti-metabolic effects on the survival and/or development of O. orientalis . Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA) were both found to significantly
185
reduce the survival and development of O. orientalis. In previous studies, transgenic plants expressing lectins have shown enhanced resistance towards a number of sap-sucking insects, including the leafhopper Nephotettix cinciteps and the planthopper Nilaparvata lugens (Gatehouse & Gatehouse 1998; Jouanin et al. 1998; Peferoen 1997). Since current chemical control agents for O. orientalis have little effect in reducing the incidence of this leafhopper and the diseases it transmits, expressing lectins with different modes of action in transgenic plants through gene pyramiding (Burrows et al ., 1999) may be an alternative strategy to provide more effective control and to combat the development of potential resistance-breaking genotypes. Several crop species have been genetically modified to express GNA and this approach has led to reduced survival of aphids and planthoppers (Chen 2008; Hilder et al . 1995; Rao et al . 1998; Shi et al . 1994; Stoger et al . 1999). As such, the genes encoding GNA and WGA might be useful transgenes in the generation of genetically modified (GM) plants with enhanced resistance to O. orientalis . Although the generation of GM crops for insect control is considered by many scientists to be a revolution to agriculture, and indeed has been widely accepted for crops not used for human consumption (such as cotton) the general public remain sceptical about the advantages and perceived risks of GM crops for human consumption. Thorough evaluation of GM crops for human consumption and public awareness/education campaigns will almost certainly be required before the technology can be implemented and/or accepted for crops such as field beans. The feeding process of sap-sucking insects takes place inside the plant tissue and is therefore difficult to characterise and study. Electrical penetration graph (EPG) is a proven method to overcome these limitations and can clearly, in real time, trace an insect’s feeding activity (McLean & Kinsey 1964; Tjallingii 1978, 1988). Although it has been successfully used for studying aphid feeding behaviour, EPG has never been adopted for O. orientalis and has only been used for a limited number of leafhopper species (Lett et al. 2001; Miranda et al. 2009; Stafford & Walker 2009). Using a combination of O. orientalis and a preferred host-plant ( Phaseolus vulgaris ), five distinctive EPG waveform patterns (O1-O5) were characterised. Although more detailed analysis of the patterns is required, together with a correlation to specific feeding activity, the results from this study provided important preliminary information regarding the feeding behaviour of O.
186
orientalis . Two waveforms, O4 and O5, which arguably represent phloem activity (salivation and ingestion, respectively), were not recorded from the artificial diet. The fact that O. orientalis can transmit TbYDV to tobacco but cannot acquire it from this plant species, suggests phloem-based resistance and an inability of the leafhopper to ingest phloem sap from tobacco, a suggestion supported by the presence of waveform O4 (salivation) and absence of O5 (phloem ingestion) when the insect was tested on tobacco. Although only preliminary in nature, the results from the electrical penetration graph opens the opportunity for further research. A better understanding of the relationships between leafhoppers with other plant species, together with the precise timing of pathogen transmission/acquisition and impact of antimetabolites on feeding, will contribute to the development of more effective control strategies.
References
Backus EA, Serrano MS & Ranger CM. 2005. Mechanisms of hopperburn: an overview of insect taxonomy, behaviour and physiology. Annual Review of Entomology 50 , 125-151.
Burrows PR, Barker ADP, Newell CA & Hamilton WDO. 1999. Plant-derived enzyme inhibitors and lectins for resistance against plant-parasitic nematodes in transgenic crops. Pesticide Science 52 , 176-183.
Chen M-S. 2008. Inducible direct plant defense against insect herbivores: A review. Insect Science 15 , 101-114.
Gatehouse AMR & Gatehouse JA. 1998. Identifying proteins with insecticidal activity: use of encoding genes to produce insect-resistant transgenic crops. Pesticide Science 52 , 165-175.
Grylls NE. 1979. Leafhopper vectors and the plant disease agents they transmit in Australia. In: Leafhopper Vectors and Plant Disease Agents (Eds K Maramorosch & KF Harris) 179–214. Academic Press, New York.
187
Harding RM & Teakle DS. 1985. Mycoplasma-like organisms as causal agents of potato purple top wilt in Queensland. Australian Journal of Agricultural Research 36 , 443-449.
Helson GAH. 1942. The leafhopper Thamnotettix argentata Evans, a vector of tobacco yellow dwarf. Journal of the Council for Scientific and Industrial Research 15 , 175-184.
Helson GAH. 1950. Yellow dwarf of tobacco in Australia V. Transmission by Orosius argentatus (Evans) to some alternative host-plants. Australian Journal of Agricultural Research 1, 144-147.
Helson GAH. 1951. The transmission of witches broom virus disease of lucerne by the common brown leafhopper, Orosius argentatus (Evans). Australian Journal of Scientific Research, Series B - Biological Science 4, 115-124.
Hilder VA, Powell KS, Gatehouse AMR, Gatehouse JA, Gatehouse LN, Shi Y, Hamilton WDO, Merryweather A, Newell CA, Timans JC, Peumans WJ, van Damme EJM & Boulter D. 1995. Expression of snowdrop lectin in transgenic tobacco plants results in added protection against aphids. Transgenic Research 4, 18-25.
Hill AV. 1941. Yellow dwarf of tobacco in Australia, II. Transmission by the jassid Thamnotettix argentata (Evans). Journal of the Council for Scientific and Industrial Research 14 , 181-186.
Hill AV. 1950. Yellow dwarf of tobacco in Australia, IV. Some host-plants of the virus. Australian Journal of Agricultural Research 1, 141-143.
Hosking JR & Danthanarayana W. 1988. Low level flight activity of Nesoclutha pallida (Evans), Orosius argentatus (Evans) and Zygina zealandica (Myers) (Hemiptera: Cicadellidae) in Southern Victoria. Journal of the Australian Entomological Society 27 , 241-249.
Jouanin L, Bonade-Bottino M, Girard C, Morrot G & Giband M. 1998. Transgenic plants for insect resistance. Plant Science 131 , 1-11.
188
Lett J-M, Granier M, Grondin M, Turpin P, Molinaro F, Chiroleu F, Peterschmitt M & Reynaud B. 2001. Electrical penetration graphs from Cicadulina mbila on maize, the fine structure of its stylet pathways and consequences for virus transmission efficiency. Entomologia Experimentalis et Applicata 101 , 93-109.
McLean DL & Kinsey MG. 1964. A technique for electronically recording aphids feeding and salivation. Nature (London) 202 , 1358-1359.
Miranda MP, Fereres A, Appezzato-da-Gloria B & Lopes JRS. 2009. Characterization of electrical penetration graphs of Bucephalogonia xanthophis , a vector of Xylella fastidiosa in citrus. Entomologia Experimentalis et Applicata 130 , 35-46.
Osmelak JA. 1986. Assessment of various insecticides for the control of the vector Orosius argentatus (Evans) (Homoptera: Cicadellidae) and tomato big bud diseases. Department of Agricultural and Rural Affairs. Research Report , 1-32.
Paddick RG & French FL. 1972. Suppression of tobacco yellow dwarf with systematic organophosphorus insecticide. Australian Journal of Experimental Agriculture and Animal Husbandry 12 , 331-334.
Paddick RG, French FL & Turner PL. 1971. Control of leafhopper-borne plant disease possibly due to direct action of systemic biocides on mycoplasmas. Plant Disease Reporter 55 , 291-293.
Peferoen M. 1997. Insect control with transgenic plants expressing Bacillus thuringiensis crystal proteins. In: Advances in Insect Control: The Role of Transgenic Plants. (Eds. N Carozzi & M Koziel)., Taylor and Francis London, 21-48.
Pilkington LJ, Gurr GM, Fletcher MJ, Nikandrow A & Elliott E. 2004. Vector status of three leafhopper species for Australian lucerne yellows phytoplasma. Australian Journal of Entomology 42 , 366-373.
Rao KV, Rathore KS, Hodges TK, Fu X, Stoger E, Sudhakar D, Williams S, Christou P, Bharathi M, Bown DP, Powell K, Spence J, Gatehouse AMR & Gatehouse JA. 1998. Expression of snowdrop lectin (GNA) in transgenic rice
189
plants confers resistance to rice brown planthopper. The Plant Journal 15 , 469- 477.
Shi Y, Wang MB, Powell KS, Van Damme E, Hilder VA, Gatehouse AMR, Boulter D & Gatehouse JA. 1994. Use of the rice sucrose synthase-1 promoter to direct phloem-specific expression of β-glucuronidase and snowdrop lectin genes in transgenic tobacco plants. Journal of Experimental Botany 45 , 623-631.
Stafford CA & Walker GP. 2009. Characterization and correlation of DC electrical penetration graph waveforms with feeding behavior of beet leafhopper, Circulifer tenellus . Entomologia Experimentalis et Applicata 130 , 113–129.
Stoger E, Williams S, Christou P, Down RE & Gatehouse JA. 1999. Expression of the insecticidal lectin from snowdrop ( Galanthus nivalis agglutinin; GNA) in transgenic wheat plants: effects on predation by the grain aphid Sitobion avenae . Molecular Breeding 5, 65-73
Thomas JE & Bowyer JW. 1980. Properties of tobacco yellow dwarf and bean summer death viruses. Phytopathology 70 , 214-217.
Tjallingii WF. 1978. Electronic recording of penetration behaviour by aphids. Entomologia Experimentalis et Applicata 24 , 721-730.
Tjallingii WF. 1988. Electrical recording of stylet penetration activities. In: Aphids, their biology, natural enemies and control (Eds AK Minks & P Harrewijn) 95-108. Elsevier, Amsterdam. van Rijswijk B, Rodoni BC, Baxter G & Moran JR. 2002. Managing tobacco yellow dwarf virus. Phase 1: Developing a diagnostic tool. Phase 2: Beginning to understand the epidemiology of TYDV. In: Department of Natural Resources and Environment pp.48. Tobacco Research and Development Corporation Final Report 1999/2001 of Project DAV 35T.
190