Vim”? .3 521.5 «1...! i, t r: if: , l . ‘ .2, .c: . 3%.. ‘ . , ‘ . ‘ . , ...n. .m.. HESlB \ um. l”‘l\‘"‘“\l 3 12\ LXMEAMQ 5 l\ \x mm, \‘ll \\ “l H Ml LIBRARY Michigan State University This is to certify that the thesis entitled An Anatomical Study of Prune Brownline Disease and Detection of Tomato Ringspot Virus in Plum presented by Karpura Kommineni has been accepted towards fulfillment of the requirements for Master's degree in Botany & Plant Pathology game! A W Major professor Date 6/4/96 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution ,_ _— ._-__- i...————.__ fi_.. 44 ._ _ .__.__.__- PLACE IN RETURN BOX to remove thle checkout from your record. TO AVOID FINES return on or betore date due. DATE DUE DATE DUE DATE DUE .i t i 0 , I ”0.:WW' AN ANATOMICAL STUDY OF PRUNE BROWN LINE DISEASE AND DETECTION OF TOMATO RINGSPOT VIRUS IN PLUM By Karpura V Kommineni A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1 996 ABSTRACT AN ANATOMICAL STUDY OF PRUNE BROWN LINE DISEASE AND DETECTION OF TOMATO RINGSPOT VIRUS IN PLUM By Karpura V Kommineni An anatomical study of bark, at the graft union of Tomato ringspot virus (TmRSV) infected, Prune Brown line diseased plum trees revealed wound areas demarcated by necrophylactic periderm. ELISA and northern hybridization were used to detect TmRSV in roots and rootstock sucker leaves of plum trees in the field. There was no significant difference between the results obtained with the two types of assays, in case of root samples. There was a significant difference in case of sucker leaf, where northern hybridization detected a higher percent infection. Chi-square analysis of the ELISA results on root or bark samples of nematode and slash inoculated ‘Stanley’lMyrobalan 29C, showed a significant difference. Silver enhanced protein-A- gold labeling of bark of the BL, scion, rootstock and sucker leaves, was distributed on cell wall and cytoplasm, with a major portion seen in the axial phloem of bark and in bundle sheath of leaf. In dedication to my father Sri K.V.Raja Rao and my aunt Smt S.Madhavi and uncle late Sri S.R.Mohana Rao iii ACKNOWLEDGMENTS I would like to thank my major professor, Dr.Donald C.Ramsdell for his support and guidance in my thesis work. I thank the members of the thesis advisory committee, Dr.Frank Ewers, Dr.Karen Klomparens, Dr.Joanne Whallon. Special thanks to Ms.Jerri Gillett for being a teacher, friend and a positive guiding spirit during the entire period of my graduate study at Michigan State University. I would like to thank Dr.Shirley Owens for her friendship and guidance. Many thanks to Dr.Richard Allison, Bill Schneider, Dr.Ann Greene for helping me with the plasmid DNA extraction and the Northern dot blot hybridization. I would also like to thank Dr.Edward Podleckis, USDA-ARS, Beltsville, MD., for his technical help throughout the project. I thank my sister, Uma Kommineni for her encouragement to pursue graduate studies and her support. I thank my husband, Kiran Sanka for his patience, support, understanding and the many discussions about my thesis work. I thank the Dept. Botany and Plant Pathology and Michigan State University for giving me the opportunity to pursue graduate studies here and broaden my horizons. TABLE OF CONTENTS List of Tables List of Figure Introduction muted Prune Brown line Disease Literature Review . A. Description of the disease 1. Early records of decline in prune trees 2. PBL can be induced by various isolates of TmRSV 3. Sources of inoculum a. Transmission by the natural vector b. Planting material c. Alternate hosts B. Virus Detection 1. Enzyme linked immunosorbent assay (ELISA) a. Reliability of ELISA for detection of woody plant viruses 2. Northern blot Hybridization a. Construction of a cDNA clone of T mRS V Materials and Methods Virus Purification Inoculation of Plum trees Nematode Inoculation for the greenhouse study Slash Inoculation for the greenhouse study Construction of the Riboprobe Miniprep Plasmid DNA Isolation Digoxigenin labeling of the riboprobe Detection of TmRSV in the plum trees Sampling Enzyme Linked Immunosorbent assay (ELISA) viii xi £503 “(A 10 12 13 15 16 16 22 23 26 29 Quantification of the virus detectable with ELISA in leaves, roots and bark tissue 30 Northern Hybridisation Extraction of Total Nucleic Acids (INA) 30 Chemiluminescent Detection with Lumi-Phos 530 31 Quantification of the viral RNA detectable with Northern hybridisation—- 32 Statistical Analysis 32 Results 1. Detection of TmRSV in the plum trees Detection of TmRSV with ELISA (Greenhouse tests) 33 Detection of TmRSV in plum trees with ELISA :Percent infection ------ 38 Quantification of the virus detectable with ELISA 42 Statistical Analysis of the ELISA results 46 Detection of TmRSV with Northern hybridization 49 II. Comparison of ELISA and Northern hybridization 60 III. Testing of plum trees from field plot in Traverse City, MI. 63 Discussion 69 Chapter I - Summary 75 Bibliography 76 M An Anatomical study of the BL and Immunolocalization of TmRSV Literature review A Hypersensitive response 80 B. Wound 82 a. Wound response 83 b. Wound healing - 84 C Types of periderm 85 D. Immunogoldlabeling 86 a. Post-embedding labeling 87 b. Immunogold labeling protocol 90 vi Materials and Methods Anatomical Study Collection of brownline samples from field plot in Traverse City -----— 92 Processing of the samples for anatomical study of the brownline ----- 92 Immunogold Labeling for light microscopy Production of T mRS VAntiserum 95 Fixation and embedding in LR White 96 Procedure for protein A-gold labeling and silver enhancement 96 Statistical Analysis of the immunolabeling data 100 Results Anatomical Study Description of a healthy graft union 101 Anatomical Study of the Brown line 101 iObservations on the Brown line area in 70031/Marianna 4001 plum -- 103 ii. Observations on the Brown line area in 70031Myrobalan 29C plum—- 105 iii. Observations on the Brown line samples from an orchard in Paw-Paw, MI 120 iv. Observations on the bark samples from the greenhouse plum trees —- 120 v.1nitial stages in the Brown line formation 123 Immunogoldlabeling ' Labeling of Brown line samples 130 Labeling of the scion and rootstock tissue of BL infected trees ----- 134 Statistical analysis of the labeling with the pre-immune serum 141 Labeling of the rootstock bark sample obtained from the greenhouse study 145 Labeling of the plum rootsock sucker leaf sample 147 Discussion ' 151 Chapter II - Summary , 162 Conclusions ‘ 163 Bibliography 165 vii LIST OF TABLES Table 1- Detection of Tomato ringspot virus in the roots of nematode inoculated plum trees with ELISA 34 Table 2- Detection of Tomato ringspot virus in the bark below the grafi union of nematode inoculated plum trees with ELISA 35 Table 3- Detection of Tomato ringspot virus in the roots of slash inoculated plum trees with ELISA 36 Table 4- Detection of Tomato ringspot virus in the bark below the graft union of slash inoculated plum trees with ELISA 37 Table 5- Detection of Tomato ringspot virus in roots and bark of plum trees with ELISA: Percent infection 39 Table 6- A chi-square analysis of the ELISA results obtained for NI root and SI root samples 47 Table 7- A chi-square analysis of the ELISA results obtained for NI bark and SI bark samples 48 Table 8- Detection of Tomato ringspot virus in the [9913 of nematode inoculated plum trees with northern dot blot hybridization 50 Table 9- Detection of Tomato ringspot virus in the bark below the grafi union of nematode inoculated plum trees with northern dot blot hybridization ------- 51 Table 10- Detection of Tomato ringspot virus in the roots of slash inoculated plum trees with northern dot blot hybridization 52 Table 11- Detection of Tomato ringspot virus in the bark below the graft union of slash inoculated plum trees with northern dot blot hybridization ----------- 53 Table 12- Comparison of ELISA and Northern hybridization relative to detected percent infection of TmRSV in plum trees - 61 viii »_' I.) Table 13- Detection of TmRSV in rootstock sucker leaf samples with ELISA and Northern hybridization: Percent infection 62 Table 14- Testing of plum trees from field plot in Traverse City, MI., for TmRSV infection. A comparison of ELISA and Northern hybridization ------------- 65 Table 15- Statistical analysis of the results obtained by testing root samples from Traverse City, MI., for TmRSV, with ELISA and Northern hybridization 66 Table 16- Statistical analysis of the results obtained by testing the rootstock sucker leaf samples from Traverse City, MI., for TmRSV, with ELISA and Northern hybridization 67 Table 17- Testing of plum trees from a field plot in Traverse City, MI. A comparison of five rootstocks relative to the percentage trees detected by ELISA and Northern hybridization 68 Table 18- Collection of brown line (BL) samples from field plot in Traverse City, MI. The field plot was surveyed for the presence of a BL in Tomato ringspot virus infected trees in November 1995 and April 1996 ------------- 93 ' Table 19- Location of the BL samples collected in the field plot in Traverse City, MI 94 Table 20- Comparison of the differences between the scions ‘7003 1 ’ and ‘Valor’ by a statistical analysis. The results obtained by surveying a field plot at Traverse City, MI., for the presence of a BL were subjected to a one way analysis of variance, in order to see if one scion is predisposed than the other to the formation of the BL 127 Table 21- Comparison of the differences between the scions ‘7003 1’ and ‘Stanley’ by a statistical analysis. The results obtained by surveying a field plot at Traverse City, MI., for the presence of a BL were subjected to a one way analysis of variance, in order to see if one scion is more predisposed than the other to the formation of the B 128 Table 22- Analysis of the immunolabeling on BL sections of a ‘70031’/Marianna 4001 tree 133 Table 23- Analysis of the immunolabeling on the scion tissue of a ‘70031’/Marianna 4001 tree 139 ix Table 24- Analysis of the immunolabeling on the rootstock tissue of a ‘70031’/ Marianna 4001 tree 140 Table 25- Analysis of the immunolabeling on the BL tissue treated with pre-immune serum 144 Table 26- Analysis of the immunolabeling on the rootstock tissue of a "Stanley’/Myrobalan 29C tree 146 Table 27- Analysis of the immunolabeling on the rootstock sucker leaf tissue --------- 150 LIST OF FIGURES Figure 1- Nematode inoculation of a ‘Stanley’/Myrobalan 29C plum tree ----------- -- 19 Figure 2- Slash inoculation of a ‘Stanley’ Myrobalan 29C plum tree with TmRSV ---- 19 Figure 3- Layout of the field plot at the North West Horticultural research station in Traverse City, MI Figure 4- Agarose gel electrophoresis of the plasmid T22 Figure 5- Collection of a bark sample from a ‘Stanley’/Myrobalan 29C plum tree for testing with ELISA and Northern hybridization Figure 6- Detection of TmRSV in roots and bark of plum trees with ELISA: Percent infection Figure 7- Quantification of TmRSV detectable with ELISA in virus-amended plum root tissue Figure 8- Quantification of TmRSV detectable with ELISA in virus-amended plum bark tissue Figure 9- Quantification of TmRSV detectable with ELISA in virus-amended plum rootstock sucker leaf sample Figure 10- Northern dot blot hybridization of total nucleic acids extracted from the roots of plum trees, sampled from a Traverse City, MI., field plot in August 1995 Figure 11- Northern dot blot hybridization of total nucleic acids extracted from the bark of slash inoculated ‘Stanley’/Myrobalan 29C plum trees, during the test conducted in February 1994 Figure 12- Quantification of TmRSV RNA detectable with Northern dot blot hybridization xi 21 25 28 41 43 44 45 55 57 59 Figure 13- A radial longitudinal section through the bark at the graft union of a healthy ‘Stanley’/Myrobalan 29C plum tree Figure 14- A plane polarized image of the healthy graft union shown in Figure 13 ----- 107 107 Figure 15- A radial longitudinal section through the graft union of a healthy ‘Stanley’/Myrobalan 29C tree 109 Figure 16- Brown line at the graft union of a PBL-affected tree in a Traverse City, ML, field plot 111 Figure 17- A radial longitudinal section through the bark at the Brown line (BL) region of a ‘70031’/Marianna 4001 plum tree 111 Figure 18- A radial longitudinal section through the bark at the BL region of a ‘70031’/Marianna 4001 tree, in an advanced stage of the wound 111 response process Figure 19- A plane polarized image of the radial longitudinal section through 113 the BL region of ‘70031’/Marianna 4001 plum tree Figure 20- A transverse section through the BL region of a ‘70031’/Marianna 115 4001 plum tree, in an early stage of wound response Figure 21- A transverse section through the BL region of a ‘70031’/Marianna 4001 plum tree, in an advanced stage of wound response ------- Figure 22- A transverse section through the BL region of ‘70031’/Myrobalan 29C plum tree, in an advanced stage of wound response ------- Figure 23- A transverse section through the BL region of ‘7003 l ’/Myrobalan 29C plum tree, in an advanced stage of wound response ------- Figure 24- A radial longitudinal view through the BL region of a ‘70031’/ Myrobalan 29C plum tree, in an advanced stage of wound ----- 115 ----- 115 ----- 115 response Figure 25- A radial longitudinal view of a part of the BL region shown 117 in Figure 24 xii 117 Figure 26- A radial longitudinal section through the BL region of a ‘70031’/ Myrobalan 29C plum tree, in an advanced stage of BL formation 119 Figure 27- A view of the boxed area in Figure 26 1 19 Figure 28- A radial longitudinal view through the BL region of a ‘70031’/ Myrobalan 29C plum tree 122 Figure 29- A view of the BL region of a 12-15 year-old plum tree from a PBL-affected orchard in Paw-Paw, MI. 122 Figure 30- A radial longitudinal section through the graft union of a ‘Stanley’ Myrobalan 29C plum tree 125 Figure 31- A higher magnification view of the graft union shown in figure 30 -------- 125 Figure 32- A plane polarized view of the graft union shown in figure 31 ------------ 125 Figure 33- A bright field transmitted view of the wound response tissue in the rootstock at the grafi union of a ‘Stanley’fMyrobalan 29C plum tree 125 Figure 34- Light microscopy immunolabeling of a brown line (BL) bark sample of a ‘Valor’/St.Julian 655-2 plum tree 132 Figure 35- Light microscopy irnunolabeling of a BL bark sample of a ‘Valor’lSt.Julian 655-2 plum tree 132 Figure 36- Light microscopy immunolabeling ofthe BL region of a ‘70031’/Mariann 4001 plum tree 132 Figure 37- Light microscopy immunolabeling of the BL region of a ‘70031’/Marianna 4001 plum tree 132 Figure 38- Light microscopy immunolabeling of the rootstock bark sample from a ‘70031’/Marianna 4001 plum tree 136 Figure 39- Light microscopy immunolabeling of the rootstock bark sample from a slash inoculated ‘Stanley’lMyrobalan 29C plum tree ------------ 136 xiii Figure 40- Light microscopy immunolabeling of the rootstock bark sample from a ‘Valor’/St.Julian 655-2 plum tree Figure 41- Light microscopy immunolabeling of the rootstock bark sample from a healthy ‘Stanley’lMyrobalan 29C plum tree Figure 42- Light microscopy immunolabeling of the scion bark sample from a ‘70031’/Marianna 4001 plum tree Figure 43- Light microscopy immunolabeling of a BL bark sample of a ‘7003 1 ’ / Marianna 4001 plum tree, labeled with pro-immune serum --------- Figure 44- Light microscopy immunolabeling of a rootstock bark sample of a healthy ‘StanleyVMyrobalan 29C plum tree, labeled with pro-immune serum Figure 45- Light microscopy immunolabeling of a rootstock sucker leaf sample of a nematode inoculated ‘Stanley’/Myrobalan 29C plum tree Figure 46- Light microscopy immunolabeling of a rootstock sucker leaf of a healthy ‘Stanley’/Myrobalan 29C plum tree xiv 136 136 138 143 143 149 149 INTRODUCTION Tomato ringspot virus (TmRSV) causes Prune Brown line (PBL) disease in prune and plum trees. The PBL disease has been reported in California (Mircetich and Hoy, 1981) and in the North Eastern United States (Brase and Parker, 1955; Cummins and Gonsalves, 1986a). Prune Brown line disease is characterized by the presence of a brown line (BL) at the graft union. The BL can be seen when the bark at the graft union is cut open, to expose the inner bark tissue. The BL is reported to be a result of necrosis of the phloem and cambial tissue (Hoy and Mircetich, 1984). Other symptoms of PBL include, chlorotic flecks on the rootstock sucker leaves, an inverted shoulder on the scion and a constricted appearance of the rootstock below the graft union. Prune Brown line has been reported to be the result of an hypersensitive reaction of a resistant scion to TmRSV infection in a susceptible rootstock (Hoy and Mircetich, 1984). Cummins and Gonsalves (1986a) have reported that PBL is analogous to Apple Union Necrosis and Decline disease in apple trees, that is also caused by TmRSV. They suggested that PBL is the result of a hypersensitive reaction of a resistant scion to TmRSV infection in a susceptible but tolerant rootstock. An anatomical study of the PBL disease has not been done so far. Tomato ringspot virus has not been localized in the bark tissue of PBL-affected plum trees, with immunolabeling. Tomato ringspot virus is a member of the nepovirus group of plant viruses. The virus has a bipartite genome of single stranded positive sense RNA (Stace-Smith, 1984). Xiphinema americanum Cobb 1913 is the natural transmission vector of the virus in Eastern North America. Cell to cell movement of the nepoviruses in the plant host has been suggested to occur through the plasmodesmata, in tubules lined with virus particles (de Zoeten and Gaard, 1969; Lucas and Gilbertson, 1994). The objectives of this research work were (1) to compare nematode. and slash inoculation treatment effects on the onset of PBL disease in ‘Stanley’lMyrobalan plum, and to determine whether natural transmission or mechanical inoculation is more effective in systemic infection and the onset of the disease. (2) To compare ELISA and northern hybridization methods of TmRSV detection to determine if one is more efficient than the other in detecting TmRSV in bark, root and rootstock sucker leaf tissue of nematode and slash inoculated trees. (3) To study the histopathological changes at the grafi union area of PBL-affected trees, in an attempt to understand the anatomical changes that occur in the early stages of the BL formation. (4) To localize TmRSV in the rootstock sucker leaf samples and the bark samples from the graft union of PBL-affected trees, so as to determine the distribution of the virus within the infected tissue. (5) To determine if there is a difference in brown line formation among five scion/five rootstock combinations in a field test planting. Chapter I Prune Brown Line Disease Literature Review A. Description of the disease Tomato Ringspot virus (TmRSV) causes Prune Brown line disease (PBL) in plum and prune trees (Cummins and Gonsalves, 1986b; Mircetich and Hoy, 1981). The PBL disease is diagnosed by the presence of a narrow strip of brown necrotic tissue, a brown line (BL), at the graft union of prune and plum trees. The brown line is a result of an hypersensitive reaction of the scion to TmRSV infection in the rootstock. The brown line is caused by the necrosis of the cambial and phloem tissues (Mircetich and Hoy, 1981). The brown line spreads around the union causing girdling, decline and eventual death of the tree (Hoy and Mircetich, 1984). The trees sometimes develop an inverted shoulder of the scion at the graft union and chlorotic spots on the leaVes of the rootstock suckers (Brase and Parker, 1955; Mircetich and Boy, 1981). The name ‘Prune Brownline’ was used in 1981 by Mircetich and Hoy. They also studied the association of TmRSV with PBL and established TmRSV as the causal agent of PBL. The disease is also referred to as 'Stanley' Constriction and Decline (SCAD) (Cummins and Gonsalves, 1986a). 1. Early records of decline in prune trees One of the early records of this condition in prune trees was made in 1955 in a study of declining 'Stanley' prune trees in New York (Brase and Parker, 1955). The 'Stanley' cultivars were propagated on Myrobalan rootstock (Prunus cerasifera Ehrh ). 4 The declining trees were distinguishable from healthy trees by the presence of a prominent inverted shoulder at the graft union and a constriction in the rootstock just below the union. In severe cases the bark at the union was necrotic and disrupted. Brase and Parker also observed chlorotic flecks on the leaves of the rootstock suckers in some of the trees. Another report of 'decline' in 'Stanley' prune trees on Myrobalan rootstock was published by Kirkpatrick et a1. (1958). The trees in this case also had symptoms similar to the above report such as constriction of rootstock below union and an inverted shoulder at the union. 2. PBL can be induced by various isolates of TmRSV Hoy and Mircetich (1984) studied the role of five isolates of TmRSV in inducing PBL. The five isolates of TmRSV associated with various diseases of Prunus spp. were peach yellow bud mosaic (PYB), cherry leaf mottle ,(CLM), California stem pitting, Prunus stem pitting and prune brownline isolates. French prunes (Prunus domestica L.) on Lovell peach, Myrobalan plum (29C) and Marianna 2624 plum rootstocks were graft inoculated at the rootstock with root chip inoculum from orchard trees that were naturally infected by the five isolates. The five isolates were readily transmitted to French prune trees on Myrobalan plum and Lovell peach rootstocks. A brown line developed at the graft union at the end of three growing seasons. Tomato ringspot virus was not transmitted to the prune trees on Marianna 2624 plum rootstock and no brownline developed in this case. 3. Sources of inoculum a. Transmission by the natural vector Tomato Ringspot virus is vectored by the dagger nematode Xiphinema americanum Cobb 1913 (Hoy and Mircetich, 1984; Teliz et al., 1966, 1967; Stace-Smith, 1984). In California, X. californicum Lamberti and Bleve-Zacheo., is the species most commonly associated with fruit trees (Hoy et al., 1984). Hoy and Mircetich (1984) demonstrated the successful transmission of TmRSV to peach and Myrobalan plum rootstocks by the TmRSV nematode vector, X californicum. However, they did not test the ability of X. californicum to transmit TmRSV to Marianna 2624 clonal rootstock. In Eastern North America, X. americanum is the most widespread species of Xiphinema (Hoy and Mircetich, 1984; Rosenberger et al., 1983; Teliz et al., 1967). However, other species of Xiphinema may be important, e.g. X. rivesi Dalmasso (F orer et al., 1984). i. Transmission of TmRSV by different life stages of X americanum The transmission of TmRSV by different life stages of X. americanum was studied by Teliz et al. (1966). The life cycle was separated into four larval stages and an adult stage. The first larval stage was the smallest in size, with the developing odontostyle in the basal portion of the functioning stylet. The second and third larval stages had the developing odontostyle enclosed in the esophageal wall posterior to the basal portion of the stylet. The fourth larval stage had the rudiments of the reproductory system defined. A mixture of larvae and adults was added at the root zone of TmRSV 111M “fl: (110. ml n2: 3r 3&3 "E 6 inoculated cucumber seedlings (800:17/pot with one seedling per pot). The nematodes were allowed an acquisition access period (AAP) of 12 days. Then the virus infected cucumber plants were cut at the soil line and healthy cucumber test seedlings were planted in the same pot. The nematodes were allowed an inoculation access period of 15 days. At the end of the IAP, the roots of the test cucumber seedlings thus inoculated with TmRSV were washed and tested by mechanical inoculation on Chenopodium amaranticolor Coste and Reyn., for symptom expression and on cucumber seedlings for serological identification by immunodiffusion assays. The first three larval stages and the . adult stage gave a 100% transmission of TmRSV. ii. Specificity of transmission Xiphinema is an ectoparasitic nematode and feeds on the roots by inserting its stylet into the roots. The apical tips of the roots are the preferred sites for feeding. The virus particles accumulate as a monolayer in the cuticle lining the lumen of the odontophore, the slender oesophagus and the oesophageal bulb (Taylor and Robertson, 1977). It has been postulated that the bound virus is released from the specific site in the nematode as a result of a pH change when the nematode salivates (McElroy, 1977; Stace- Smith and Ramsdell, 1987). An hypothesis has been proposed that nepovirus transmission is linked to the interaction between the vector and the viral protein coat. The specificity of transmission reflects the specific association between the protein surface of the virus and the cuticle at the site of retention within the nematode vector. The less serologically related the viruses, the more unlikely it is that the same nematode will vector them (McElroy, 1977). 1 P11 01 n can time ' I 7.531 U63 (‘1‘! b. Planting Material When virus-infected rootstocks are used in propagation they can serve as a source of inoculum and cause PBL in the trees. Mircetich and Hoy (1981) studied the French cultivar 'Agen' of prune on Myrobalan plum rootstock in California. They showed graft transmissibility of TmRSV fi'om PBL-affected peach and Myrobalan plum seedling to healthy ‘Lovell’ peach rootstock and Myrobalan plum seedlings, and to French prune trees on ‘Lovell’ peach and Myrobalan plum rootstocks. Root chips from PBL infected peach trees when grafted onto the rootstock or scion of French prune trees resulted in transmission of the virus to the rootstock and not to the scion. Grafting with buds from the scion of PBL-infected trees did not result in transmission of TmRSV to the rootstock or scion. Tomato ringspot virus was not detected by enzyme linked immunosorbent assay (ELISA) from the scion portion of either the experimental trees or the PBL-affected trees in the orchards (Mircetich and Boy, 1981). i. Susceptibility of various rootstocks of Prunus to PBL Based upon field observations, Mircetich and Hoy (1981) suggested that a clonal rootstock, Marianna 2624 (P. cerasifera x P.munsonia[?] Wight & Hedr.), may be more resistant to PBL than peach or Myrobalan plum rootstocks. Hoy and Mircetich, (1984), in a study comparing peach (P. persica[L.] Batsch), Myrobalan 29C plum and Marianna 2624 plum rootstocks, found Marianna 2624 to be resistant to TmRSV infection when challenged with root chip inoculmn from PBL-affected orchard trees. 01 ill (L ,.. 8 Cummins and Gonsalves (1986b) studied a pool of PBL-affected trees propagated on the following rootstocks: P. domestica cvs. Brompton, Yellow Egg and St. Julian A; P. cerasifera Ehr. cv. Myrobalan B; and five Myrobalan seedling lines. A Chi-square analysis revealed no differences among rootstocks for incidence of infection or for lateral distribution of infection within an individual tree. ii. Are some cultivars more susceptible? Brase and Parker (1955), in a comparison of three prune cultivars, Fellenberg, Stanley and Abundance, propagated on Myrobalan seedling rootstock with chlorotic fleck, found that constriction of the rootstock below the union was seen on the Stanley cultivar and not on the other two cultivars. Another symptom observed was alternating long ridges and depressions on the rootstock immediately below the union resulting in an appearance of angularity in cross section. The authors suggested that there may be some factor in Stanley that causes the angularity of the stock without serious injury. They also suggested that when this factor combines with the one that causes chlorotic fleck, a constriction is formed at the bud union, roots develop poorly and some of the tree's die. c. Alternate Hosts Common orchard weeds also serve as resorvoirs of TmRSV. In a cooperative field study involving apple and peach orchard sites in Pennsylvania, New York and Indiana, it was found that 21 weed species in 12 families were infected with TmRSV in one or more locations. Some of the common weeds that were TmRSV-positive were herbaceous annuals e.g. the common chickweed (Stellaria media (L.)Villars.) and lamb's quarter (Chenopodium album L.), herbaceous perennials such as the dandelion (Taraxacum (Wirint 11118103 ““16 hush l 1‘ teeth} B. lit 1. [an Ll Width 1988). .etho-d misty enzyme sap b} intensit ConlUg: met 0d be bou ‘illh [h Mice. 9 oflicinale Weber), broadleaf plantain (Plantago major L.) and sheep sorrel (Rumex acetosella L.) (Powell et al., 1984; Stace-Smith and Ramsdell, 1987). The TmRSV- infected weeds serve as a source of virus inoculum for the nematodes to transmit to healthy commercially important trees in the orchards. B. Virus Detection 1. Enzyme linked Immunosorbent Assay The enzyme-linked-immunosorbent-antibody assay (ELISA) is by far the most widely used serological diagnostic tool in plant virology (Clark, 1981; Miller and Martin, 1988). The direct Double Antibody Sandwich (DAS)-ELISA is the commonly used method of ELISA, in which untagged antibody is bound to a solid phase, e.g. wells of a polystyrene microtiter plate. The test sample, enzyme-conjugated-antibody, and the enzyme substrate are added sequentially, while unbound material is removed after each step by washing. In a positive test, the substrate solution becomes colored, and the intensity of the color, determined spectrophotometrically, is proportional to the amount of conjugate bound and, consequently, to the amount of antigen present. In a variation of the method, indirect ELISA (I-ELISA), the specific primary antibody is used untagged and the bound antibody is detected using an enzyme-conj ugated secondary antibody reactive with the irnmunoglobulin species of the primary antibody (Miller and Martin, 1988). Reduced specificity for detecting strains of Tobacco mosaic virus (TMV) was found using this procedure (Clark, 1981). 1.1 1 in ii] deal I00- 10 a. Reliabilty of ELISA for detection of woody plant viruses ELISA has been used for the detection of woody plant viruses in different kinds of plant samples e.g. leaf, root and bark. The sensitive detection of the virus by ELISA is dependent on the quality of the antisera available, the plant part sampled and its condition, the titer of the virus in the sample, the time of sampling and where on the plant the sample was taken. i. Titer of virus and uneven distribution in the tree Even in instances where the visualsymptoms of the disease have progressed to an advanced stage, it may be difficult to detect the virus in Woody plants. In apple trees with Apple Union Necrosis and Decline (AUND) disease caused by TmRSV, when the inner bark of the rootstock from trees with the graft union symptoms was sampled, TmRSV was not found in 11% of the trees (Rosenberger et al., 1983). The failure to detect TmRSV was probably due to uneven distribution of virus within rootstock, due to low virus titer, or due to interfering compounds in the tissue. ii. The season when the test is done The time of the year when the sampling is done is also important. TmRSV was detected in the leaves of the scion of orchard peach trees in May and in the roots and bark in July (Bitterlin et al., 1984). In another study, TmRSV was more efficiently detected in the leaves of peach and apple seedlings after a period of dormancy (F orer et al., 1984). The authors suggested that the dormancy induced the movement of the virus from the roots to the leaves. it Site 15cm ; detect: ifCIT. tn iexx 11 iii. Site of sampling on the tree Tomato ringspot virus was detected in 100% of bark samples by I-ELISA when samples were taken directly below the brownline. The BL on the affected trees was seen on just One side of the circumference of the tree, and when samples were taken up to 15cm around the circumference of the tree from the end of the brownline, TmRSV was detected in only 28% of the trees. No TmRSV was detected in 56% of the samples taken >15cm from the end of the brownline (Hoy and Mircetich, 1984). The importance of sampling at the optimal place on the tree is also illustrated by a review of a study of Prunus stem pitting (PSP) disease by Bitterlin et a1. (1988). Bark samples taken from the lower part of the trunk of peach trees were the most reliable tissue source for detecting the virus in infected orchard trees. The highest percentage of ELISA- positive samples were obtained from samples taken within the first 10 cm below the soil line. Sometimes ELISA-positive and ELISA-negative samples were located within 1 to 2 cm of each other. In a majority of the PSP-affected trees surveyed in this study, the graft union was not clearly discernable and coincided with the soil line in some trees. Hence, the position of the sampling is given relative to the soil line. The virus was not detected in any leaf or bark samples of the upper branches. However, TmRSV was detected in the scion bark samples taken just above the soil line. In the case of root samples, virus detection was lower in the distal portions than in the proximal portions of the tree roots. 111 0‘,» 1 U 12 iv. Quantity of the sample Cummins and Gonsalves (1986b), in a study of PBL on 'Stanley' trees, suggested that bark samples at the graft union area be taken from at least three of the four quadrants of the trunk in order to insure detection of the virus by ELISA . v. Type of sample assayed for the virus Extracts from rapidly growing shoot tip leaves of healthy apple trees contain antigens that can produce an ELISA positive result (false positive) for a TmRSV assay. The unidentified antigens also formed precipitates in agar gel double diffusion tests. The authors suggested that apple shoot tips should not be tested for regulatory purposes and virus certification programs (Mink et al., 1985). 2. Northern blot Hybridization Other techniques, e. g. nucleic acid hybridization can be used for detection of plant viruses. In nucleic acid hybridization, viral nucleic acid probes are used to hybridize with homologous viral nucleic acid sequences in a dot blot assay. When RNA probes are used in nucleic acid hybridization, the technique is called Northern hybridization. The probes for plant viruses are usually complimentary DNA (cDNA) probes, since most plant viruses have an RNA genome (Miller and Martin, 1988). Transcription vectors carrying promoters specific to either Bacteriophage SP6 or Bacteriophage T7 and T3 have in f R\' D\ in I b) rib Pd l3 facilitated in vitro transcription of cloned DNA sequences when the bacteriophage- specific RNA polymerase is added to the reaction (Maniatis et al., 1982; Melton et al., 1984). Thus, ssRNA probes can be generated which are more effective in northern hybridization than nick-translated DNA probes. The RNA probes make possible RNAzDNA or RNA:RNA hybridization. The RNA:RNA duplexes are more. stable than DNAzDNA or DNAzRNA duplexes. This stability allows for more stringent washing conditions of the hybrids, increasing the signal to noise ratio. There is a further advantage in that the unbound RNA and the nonspecifically bound RNA on the blot can be removed by RNase digestion. However, there is a possibility of the RNA probe binding to ribosomal RNA. Unbound or non-duplexed RNA probes also have susceptibility to RNase digestion (Melton et al., 1984). The detection of the nucleic acid hybrid is made possible by labeling the probe with a radioactive or nonradioactive label. Some of the nonradioactive labels used are biotin-avidin conjugated to alkaline phosphatase or horse radishperoxidase (Miller and Martin, 1988), and digoxigenin that can be detected by a colorimetric or chemiluminescent method (Boehringer Manheim Corp., 1994). a. Construction of a cDNA clone of T mRS V A TmRSV cDNA clone comprised of regions of RNA-1 and RNA-2 of a TmRSV isolate associated with Prunus stem pitting disease and AUND in the eastern United States was constructed by Hadidi et al. (1989). This clone was inserted into the polylinker region of the SP6 transcription vector pSP64. Using the SP6 promoter flanking this region, high specific activity RNA probes can be generated by SP6 RNA polymerase. 14 The RNA probe can be used for detection of TmRSV in northern blot hybridization assays. As little as 1pg of TmRSV RNA was detected in a dot blot assay with a probe generated in the above manner (Hadidi and Hammond, 1989). i. Problems in detection Tomato ringspot virus has been detected in nectarine (Prunus persica [L.] Batsch) trees using transcribed RNA probes specific to RNA-2 of the virus (Powell et al., 1991). Extracts from bark removed from areas of the tree where stem pitting symptoms occurred contained detectable TmRSV RNA. Extracts from bark removed from above the stem ‘ pitted region or fiom the opposite side of the trunk, where symptoms were absent, did not contain any detectable RNA. The authors found hybridization to be. more efficient than ELISA in detecting TmRSV from bark samples. However, ELISA was more efficient when root samples were tested. The reason for this result is not known. Vii 7‘1 1 C01 we Ema 10 V0 Ch b} Materials and Methods Virus Purification Tomato Ringspot virus was propagated in ‘National Pickling’ cucumber seedlings. The TmRSV-infected cucumber tissue was homogenized in a cold Waring blender with cold 0.5M borate buffer (0.05M borax, 0.2M boric acid), pH 7.4 (1:2 w/v). The extract was filtered through four layers of cheese cloth, fiozen and slow thawed at 4°C. All futher steps were performed at 4°C. A low speed centrifugation was done at 10,000 rpm for 20 min. Ammonium sulphate at a concentration of 15 g/ 1 00ml was added to the supernatant and the virus precipitated overnight. After a low (10,000 rpm for 15 min) and high speed centrifugation (28,000 rpm for 2.5 hrs), the resultant pellet was resuspended in 0.05M phosphate buffer, pH 7.5. The virus was purified through [5 to 30% (w/v)] linear-log sucrose density gradients in an SW41 rotor at 38,0001'pm for 90 min. The gradients were fractionated on an ISCO Fractionater. The middle and bottom components were collected. The virus was pelleted by a high speed centrifugation at 28,000rpm for 6.5 hr and the pellet was resuspended in 0.05M phosphate buffer. Extraction of TmRSVRNA The purified virus particle suspension in 0.05M phosphate buffer was mixed with 10% SDS (sodium dodecyl sulphate), for a final concentration of 1% SDS, and gently vortexed for 2 min at room temperature. Phenol-chloroform extraction Was done twice on this mixture, and the virus was precipitated with 3M sodium acetate (0.1 vol), pH 5.0, and chilled ethanol (3 vol). The mixture was kept at -20°C for 2 hr and the viral RNA pelleted by spinning at 14,000g for 15 min. The pellet was resuspended in TE buffer (IOmM Tris 15 3:11 In mm @1011 atria Pickling nil-gm: 0031661 Xiphim 1111}; SUCH] [TEES V 16 and lmM EDTA), pH 8.0. The concentration of the RNA was determined with an uv spectrOphotometer by taking O.D readings at 260nm. The purity of the RNA was determined by taking readings at 260nm and 280nm. Inoculation of Plum Trees Nematode Inoculation for the greenhouse study Twenty five 1-year old ‘Stanley’/Myrobalan 29C plum trees were planted in five gallon plastic pots in sterilized soil, in August 1993. The nematode vector Xiphinema americanum was added to the soil in the root zone at the rate of 1000:15 per pot. ‘National Pickling’ cucumber seedlings at the rate of 5-6 per pot were planted in the same pots (Figure 1). The cucumber plants were mechanically inoculated with purified TmRSV at the cotyledon leaf stage and served as source of viral inoculum for the nematodes. As a control Xiphinema americanum was added to the soil of five ‘Stanley’ plum trees at the rate of 1000:15 per pot, and healthy cucumber seedlings served as the bait plants in this group. The plants were maintained in the greenhouse for the entire duration of the study except when they were kept in cold storage to undergo dormancy for 40 days in the winter. Slash Inoculation for the greenhouse study Another group of 25 'Stanley'/Myrobalan 29C plum trees were slash inoculated, in September 1993, with purified TmRSV at and a little below the graft union. Sterile razor blades were used to make cuts on the bark and simultaneously a solution of TmRSV in sucrose was dripped with a pasteur pipette on the cut areas (Figure 2). As a control, 10 plum trees were inoculated with sucrose solution in the same way as described above. This group ~IA .11. lilt~ Int 1V} 1 m1 1 7 of plants was maintained in the greenhouse and treated in a manner similar to the nematode inoculated trees above. Inoculation of plum trees in afield plot in Traverse City, MI. A field plot was set up at the regional NorthWest Horticultural Research station in Traverse City, MI, in the summer of 1993. The plot was a completely randomised design of l-year-old plum cultivars of five scions and five rootstocks (Figure 3). The plum rootstocks were Myrobalan 29C, Marianna 4001, Marianna 2624, Marianna GF8-1 and St. Julian 655- 2. The scion cultivars used were Stanley, New York 5890012, 70031, Valor and Carolyn Hanis. Another plot, identical to the above was also planted adjacent, and served as the control for the study. The plum trees in the experimental plot were nematode-inoculated with the vector, X iphinema americanum, and slash inoculated with purified TmRSV in the manner detailed above. 18 Figure 1. Nematode inoculation of a ‘Stanley’/Myrobalan 29C plum tree. Cucumber plants, 5 to 6 in number, were planted around the plum tree and inoculated with TmRSV. Nematodes added to the soil, fed on the roots of the TmRSV infected cucumber plants, acquired the virus and transmitted it to the roots of the plum tree. Figure 2. Slash inoculation of a ‘Stanley’/Myrobalan 29C tree with TmRSV. Cuts were made on the bark, at and below the graft union of the plum tree with a razor blade. Simultaneously a solution of TmSRV was dripped onto the slash injuries with a pasteur pipette, thus inoculating the tree with TmRSV. 20 Figure 3. Layout of the field plot at the North West Horticultural research station in Traverse city, MI. The plot was a completely randomized design of five scions and five rootstocks. The plum rootstocks are Myrobalan 29C (Myro 29C), Marianna 4001 (Mar 4001), Marianna GF 8-1 (Mar GF8-1), Marianna 2624 (Mar 2624) and St.Julian 655-2 (St Jul 655-2). The scion cultivars are Stanley (St), New York 58.900.12. (NY), 70031, Valor 01 a1) and Carolyn Harris (CH). There were two plots such as the one shown here. The southernmost plot was inoculated with TmRSV and the northern plot was a non- inoculated control. ~ 0 C C C .— _.. C C O— _. O I O .— Wmmnm.hmmmm mmnmm nmmm.m r5222 U r2362; U mete; U lla.n35.t. J _.Heeee__..e..eeee__eeeee__eeoeo_ mmamw Wmmam Mmmmn mammw roouofiz U CASSEU _l_8exm¢Eh .«o 55339 .0 enema 42 Quantification of the virus detectable with ELISA The result for the quantification of TmRSV detectable with ELISA in virus-amended plum root tissue is shown in Figure 7. The least detectable quantity of the virus was 24ng/well with an A 405 nm value of 0.243. The least detectable amount of the virus detectable in virus-amended plum bark tissue was 20ng/well with an A 405 nm value of 0.363 (Figure 8). The least detectable quantity of TmRSV in the virus-amended rootstock leaf was 39ng/well with an A 405 nm value of 0.602.(Figure 9). N‘ :3: wcv 382.6 2:5 Co EzoE< 2.0 050 m we we 02 omw oomwr ills:f1.l,IrllI.ll|lel1111.1lltIIlIlkll-llvlrtvtllllll 03mm: Loom Esr— coceoEaAE? E «aw—Am 5; 2253.23 >mmEhLo cotauczcszO .5 3:3... _ . oooom o No v.0 mo mo we we we (run-W“ v) oaurrqrosqv Ems/mi 382% WEI/B EsoE< mmoo mfo 0.0 VN mm mm one mmo comm ooooe 44 . _ . _ o _ ............. Lilllll II. II 1|..-111Lll.» Ill_llllnllll.rrlitlrllilllhllltiuillI2.r...;. :1 . . _ . . . . _ ,. _ . _ . No _ ..---. .-..,...-1», .. : v.0 ......... .............. . - - L . mo ma lllllllll me Ill!“ lllllllllllllllllllll . 111111 , iiiiiiiiii 1.111 m—x _ _ l . . . u . _ .. . . osmmz 1.5m Es: coceofinézt.» E («m—4m :23 325886 Lama—ch? 53352.30 .m 2:3... Huugm- V) munqrnsqy Eu: mi 02022.5 3:.) mo E3252. 500.0 3.0 00 em 0.0 mm 02 0N0 000m 0000f . . ., . w _. r o m0 v0 00 0.0 .\. we we 0e 0e . 22mm: .23— .5225 zoowfleom Eza 30585-22? E {wing at», «38830 army—EH? :ocmoczzwso .o 2:07.— (LUU""V) aoueqrosqv 46 Statistical Analysis of the ELISA results Ch i-square analysis for differences between treatments A chi-square analysis (Table 6) of the ELISA results for NI root and SI root was done. A chi-square analysis (Table 7) of the ELISA results obtained in case of NI bark and SI bark was also done. The chi-square analysis in both cases revealed a significant difference between the two treatment effects on plum trees in their response to TmRSV infection. 47 Table 6. A chi-square analysis of the ELISA results obtained for NI root and SI root samples. W: There is no difference between nematode inoculation and slash inoculation treatment effects on plum tree roots in their response to TmRSV infection, and any differences in results are due to chance alone. Treatment type TmRSV Infected Uninfected Total NI Root" Observed (O) 8 17 25 Expected (E) (14.5) (10.5) (25) SI Root° Observed (O) 21 ' 4 25 Expected (E) (14.5) (10.5) (25) Total 29 21 50 ‘: Data from Table 1 °: Data from Table 3 Expected infected = 29 / 50 x 25 =14.5 Expected uninfected = 21 / 50 x 25 ~ = 10.5 Chi-square value with Yates correction = [(O-E)-0.5]2 / E = 11.83 The chi-square value is comparable to the chi-square table value with a probability of 0.001 of obtaining a value as large or larger than 10.827. Therefore the chi-square value is significant and the null hypothesis is rejected. Conclusion: There is a significant difference between nematode and slash inoculation treatment effects on plum tree roots in their response to TmRSV infection. Table smpl Null 1 new and 21 Exp Chi 48 Table 7. A chi-square analysis of the ELISA results obtained for NI bark and SI bark samples. ‘ ' anLHypothosis: There is no difference between nematode inoculation and slash inoculation treatments effects on plum tree bark in their response to TmRSV infection, and any differences in results are due to chance alone. Treatment type TmRSV Infected Uninfected Total NI Barka Observed (O) 12 13 25 Expected (E) (17) (8) (25) SI Bark° Observed (O) 22 3 25 Expected (E) (17) (8) (25) Total 34 16 50 8: Data from Table 2 °: Data from Table 4 Expected infected = 34/50 x 25 =17 Expected uninfected = 16 / 50 x 25 =8 Chi-square value with Yates correction = [(O-E)-0.5]2 / E = 7.4448 The chi-square value falls between the chi-square table values of 6.635 and 10.827 with probabilities of 0.01 and 0.001, that the results are due to chance alone. Therefore the chi- square value is significant and the null hypothesis is rejected. Conclusion: There is significant difference between nematode and slash inoculation treatment effects on plum tree bark in their response to TmRSV infection. 49 Detection of TmRS V with Northern H ybridiZation There was 52% infection by TmRSV in the roots (Table 8) and 24% infection by TmRSV in the bark (Table 9) of nematode inoculated trees at the end of six tests, as detected with northern hybridization. There was 48% infection by TmRSV in the roots (Table 10) and 12% infection by TmRSV in the bark (Table 11) of slash inoculated trees. A result of the northern dot blot hybridization of total nucleic acids extracted from the roots of plum trees sampled from a field plot in Traverse City , MI., is shown in Figure 10. Note that the positive signal for the total nucleic acids blotted on the membrane, is in the form of an open circle (arrow). A result for the northern dot blot assay of total nucleic acids, extracted from the bark of slash inoculated ‘Stanley’/Myrobalan 29C trees during the test conducted in February 1994, is shown in Figure 11. Quantification of TmRS VRNA detectable with Northern Hybridization All amounts of the viral RNA dot blotted, i.e. 50ng - 0.6pg/spot, were detectable with northern hybridization (Figure 12). Note that the spots of the viral RNA are a full circle in contrast to the pattern obtained when total nucleic acids from the roots were dot blotted. Tab? 1166i he. _ _ _ _ :Fn EL \\KN\\\\K\\\\\\T\%\ «U. Tl. Te: TEf 50 Table 8. Detection of Tomato ringspot virus in the roots of nematode inoculated plum trees with northern dot blot hybridisation Positive Tree No Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 at least 09- ‘93 02- ‘94 05- ‘94 09- ‘94 02- ‘95 07- ‘95 one time - + + + 1 2 3 4 5 6 7 8 9 ++++++++- ++ Total Percent 12 y—n N Ul N Test 1 = Test conducted in November 1993 Test 2 = Test conducted in February 1994 Test 3 = Test conducted in May 1994 Test 4 = Test conducted in September 1994 Test 5 = Test conducted in February 1995 Test 6 = Test conducted in July 1995 ‘ Percent infection, as a percentage of 25 trees tested. lab! nem he (I EL/l/T/l/l/l/llllllIjll7_rllT 51 Table 9. Detection of Tomato ringspot virus in the bark below the graft union of nematode inoculated plum trees with northern dot blot hybridisation. Positive Tree No Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 at least 11- ‘93 02- ‘94 05- ‘94 09- ‘94 02- ‘95 07- ‘95 one time 1 2 3 4 5 6 7 8 9 Test 1 = Test conducted in November 1993 Test 2 = Test conducted in February 1994 Test 3 = Test conducted in May 1994 Test 4 = Test conducted in September 1994 Test 5 = Test conducted in February 1995 Test 6 = Test conducted in July 1995 a Percent infection, as a percentage of 25 plum trees tested. 52 Table 10. Detection of Tomato ringspot virus in the roots of slash inoculated plum trees with northern dot blot hybridization. Positive Tree No Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 at least 11- ‘93 02- ‘94 05- ‘94 09- ‘94 02- ‘95 07- ‘95 one time + + + 1 2 3 4 5 6 7 8 9 Total Percent Test 1 = Test conducted in November 1993 Test 2 =Test conducted in February 1994 Test 3 = Test conducted in May 1994 Test 4 = Test conducted in September 1994 Test 5 = Test conducted in February 1995 Test 6 = Test conducted in July 1995 a Percent infection, as a percentage of 25 plum trees tested. Tel no. it: _ r n K Rim \ n \ N N \ N \ \ \ \ \ K \1