UNIVERSIDADE DO ALGARVE UNIDADE DE CIÊNCIAS E TECNOLOGIAS AGRÁRIAS Interrelationship between two variants of the Circulifer tenellus complex and Spiroplasma citri the causal agent of stubborn disease Margarida Lynette S. Solano d' Almeida FARO 1992 gq-lZ UNIVERSIDADE DO ALGARVE UNIDADE DE CIÊNCIAS E TECNOLOGIAS AGRARIAS Interrelationship between two variants of the Círculifer tenellus complex and Spiroplasma citri the causa! agent of síubborn disease Margarida Lyneíte S. Solano d' Almeida Dissertação apresentada na Universidade do Algarve para efeito de prestação de Provas de doutoramento. FARO 1992 ?s //92V ALtíJ-rJ: 1 AC KNOWLE DGMENTS Firstly I wish to thank Professor Benjamim Raccah and Professor Meir Klein of the Plant Protection Department of the Volcani Center in Israel, for their advise, supervision and encouragment and for giving me the opportunity to carry out this work. I am grateful to ali the staff of the Virology Department of the Volcani Center for their help, advise and friendship and giving me the strengh to carry on. I also wish to thank Doctor Abdullah Gera and Doctor Amit Gal-On of the Volvani Center for ali their kindness and help. I am especially grateful to the University of Algarve, in the person of the Dean, Professor Lloyd Braga, for having granted a scholarship to do this work in Israel and to his successor Professor Montalvão Marques and Professor Eugénio Faria who authorized its renewal until its completion. A very special thanks to Professor Lorete Anunciada for ali her support, advise and friendship. To Ms. Ercilia Cavaco a thank you for her help in the final stages of assembling this thesis. And iast but not least I would like to say thank you to my colleagues Maria João Correia, Manuela David and Filomena Fonseca who in addition to their own duties substituted for me at the University for nearly four years. ±7\ r> O mT>J- X\X 7\ mX Stubborn disease causes important economic lesses in most of the citrus growing regions, namely in the Mediterrranean area. Its causal agent is a mycoplasma-like-organism (MLO), Spiroplasma citri, and its vectors are leafhoppers of the Circulifer/Neoaliturus complex. Two variants of the Circulifer tenellus complex were found in "stubborn" affected areas, in Israel, Circulifer tenellus-A on Atriplex halirnus and Circulifer tenellus-P on Portulaca oleracea, in the Jordan Valley and coastal plain, respectively. A study was carried out in order to characterize the two leafhoppers based on morphology, host preference and vectorial capability. Morphologically they were similar, except for color and the presence of a denticle on the 7th sternum of the female. Host preference was quantified by the longevity and fecundity on several host piants. Both leafhoppers were found to prefer Cruciferae and Chenopodiaceae but differences were observed between the two leafhoppers, namely with Brassica oleracea, Brassica rapa, Beta vulqaris and Matthiola inçaria. The latter was the most suitable and chosen as the host plant to maintain the cultures and for transmission studies. The rate of transmission by Ç. tenellus P after being injected with a Spiroplasma citri isolate v/as 5.0%, but when the pathegen was acquired by feeding it was 2.6%. With the variant C.. tenellus A, the rate of transmission was 42.2% and 11.9% respectively. An effect of the pathogen on the longevity and fecundity of the variant Ç. tenellus-A was observed. Cresses between the two variants did not produce descendents. Attempts were also rnade to characterize S_. citri isolates found in different geographical regions of Israel. Serological and electrophoretic methods and DNA-DNA hybridization were used. Isolates appeared to be similar, however, those collected from Valencia orange in the coastal plain, showed differences when a Western blot technique was used. RESUMO A doença "stubborn" causa perdas económicas importantes na maioria das regiões citrícolas, nomeadamente na area Mediterrânea. 0 agente causal, Spiroplasma citri, e um "mycoplasma-like-organism" (MLO) e os seus vectores são cicadelídeos do complexo Circulifer/Neoaliturus. Foram encontradas duas variantes do complexo Circulifer tenellus em areas afectadas pela doença "stubborn", em Israel, uma Ç. tenellus-A colectada em Atriplex halimus e outra, Ç. tenellus-P em Portulaca oleracea, no Vale do Jordão e na planície costeira, respectivamente. Foi realizado um estudo de modo a caracterizar os dois cicadelideos, baseado na morfologia, preferencia de hospedeiro e capacidade vectorial. Morfologicamente as duas variantes sao similares, excepto na cor e na. presença de um denticlo no sétimo esternito da fêmea. A preferencia de hospedeiro foi quantificada através da longevidade e fecundidade em diferentes plantas hospedeiras. Verificou-se que os dois cicadelídeos tinham preferência por plantas das famílias Cruciferae e Chenopodiaceae embora se tenham observado diferenças, nomeadamente com as especies botânicas Brassica olearacea, Brassica rapa, Beta vulgaris e Matthiola incana. Esta última foi escolhida para manutenção das culturas e experiências de transmissão. A taxa de transmissão por Ç.. tenellus-P após ter sido injectada com _S. citri foi de 5.0% e 2.6% após aquisição do agente patogenico por injestáo. Cora a variante Ç. tenellus-A a taxa de transmissão foi de 42.2% e 11.9% respectivamente. Verificou-se que S.. citri afectava a longevidade e fecundidade de Ç. tenellus-A. Cruzamentos entre os dois cicadelídeos não produziram descendentes. Foi também feito um estudo preliminar a fim de caracterizar isolamentos de S_. citri colhidos em diferentes regiões de Israel, utilizando métodos serologicos, analise de proteinas por tílec Lrophortiae e uxb idi z-ciucuj dt; DNAa . Apenas um isoiamenro, extraído de laranja Valencia colhida a planície costeira, apresentou diferenças quando a técnica de "Western blot" foi aplicada. V INDEX INTRODUCTION 1 LITERATURE REVIEW The leafhopper 4 - Distribution 4 - Host Plants 6 - Cultivated Plants 7 - Number of generations 7 - Migration and dissemination of C. tenellus 8 Biology of C. tenellus - Reproduction 9 - Preoviposition 10 - Oviposition 10 - Nymphal development 10 - Citrus stubborn disease - Distribution in the U.S. A 11 - Distribution in the Mediterranean area 11 - The causal agent of stubborn - Biology cf Spiroplasmas - Classificatioin and Phylogeny 13 - Structure and Function 14 - Transmission of S. citri - Vectors in the U.S.A 15 - Vectors in the Mediterranean countries 16 - Hosts of Citrus Stubborn Disease Pathogen 17 - vecror - patnogen Keia-cionsnips - Transmissibility 18 - Pathogenicity 20 - Spiroplasmas - Identification 21 MATERIAL AND METHODS 1. Climatic conditions in Israel. Citrus areas affected by stubborn disease 23 2. Insects - Collection 27 3. Cages 3.1. Rearing cages 27 3.2. Cages used for transmission experiments 29 3.3. Mating and oviposition cages 30 4. Isolation of Spiroplasma citri 31 4.1. Isolation procedure 31 4.2. Culture médium 32 5. Enzyme-linked Immunosorbent Assay (ELISA) 33 5.1. Preparation of antiserum against S.. citri 35 5.2. Purification of -globulin (IgG) 35 5.3. Conjugation of Alkaline Phosphatase with IgG 36 CKAPTER I - BIOLOGY OF THE BEET LEAFHOPPER COMPLEX 1. General characteristics of Circulifer tenellus (Baker) (Homoptera: Cicadellidae) 37 2. Description of two variants of the Circulifer tenellus complex 41 Vil i tt „ _ 1 _ 4- (: 4_ V-. x~. .r, r~* : — 1 ; c ._, J . llGSll — WJ_ ^11>- ovvv> V,.1--L'^1-ÍX-L.1-C:-L Utz:il»=:X X Ufa variants 3.1. Introduction 41 3.2. Methods 3.2.1. Insects 42 3.2.2. Plants 42 3.3. Procedure 3.3.1. Experiment 1 43 3.3.2. Experiment 2 43 3.3.3. Experiment 3 43 3.3.4. Experiment 4 44 3.4. Results 44 4. Life cycle of two variants of the Circulifer tenellus complex reared on Matthiola incana 4.1. Introduction 51 4.2. Kethods 51 4.3. Procedure 51 4.4. Results 52 5. Crossbreeding two variants of leafhoppers from the Circulifer tenellus complex 52 5.1. Procedure 5.1.1. Experiment 1 52 5.1.2. Experiment 2 53 5.1.3. Experiment 3 53 5.2. Results 53 CHAPTER II - TRANSMISSION OF Spiroplasma citri 3Y TWO VARIANTS OF THE Circulifer tenellus COMPLEX 1. Introduction 55 2. General methods adopted in S.. citri transmission experiments 55 2.1. Injection of disease agents 55 Vil: 2.2. AcquitiiLiun dcutitjíá feediuy (AAr ) un infected plants 55 2.3. Incubation of the disease agent in the leafhopper. 55 2.4. Inoculation access feeding 56 2.5. Monitoring infection in plants exposed to infective leafhoppers 56 3. Inoculation of leafhoppers by injection of four isolates of S_. citri 3.1. Methods 56 3.2. Transmission by leafhoppers which acquired S. citri by feeding 5 7 3.3. Results 57 4. Vector - pathogen relationships 4.1. Determination of minimal Acquisition Access Period (AAP) 65 4.1.1. Methods 65 4.1.2. Results 65 4.2. Transmissibility by males and females of variant Ç. tenellus-A 6 9 4.3. Incubation Period (IP) 4.3.1. Methods 70 4.3.2. Results 70 4.4. Inoculation Access Period (IAP) 72 4.4.1 Methods 7 2 4.4.2 Results 72 4.5. Relation between presence of S. citri in an infected plant and rate of transmission by the vector Ç. tenellus-A 4.5.1. Introduction 75 4.5.2. Procedure A. Application of ELISA to the detection of .. 75 S. citri in olants 1 /'V 4.5.3. Results 76 5. Pathogenicity of S.. citri by Ç. tenellus-A 5.1. Iritroduction 78 5.2. Procedure 7 8 5.3. Results 5.3.1. Effect of S.. citri on the longevity of C. tenellus-A 7 9 5.3.2. Effect of S. citri on fecundity of C. tenellus-A 81 CHAPTER III - CHARACTERISTICS OF S. citri ISOLATES 1. Serological relatedness of Spiroplasmas by Enzyme-linked Immunosorbent Assay (ELISA) 84 1.1.