Doctoral Degrees (Plant Pathology)

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Application of transmissible agents to control citrus tree size.
(1997) Van Vuuren, Stephanus Petrus.; Da Graca, John Vincent.
Establishing citrus orchards at higher densities has become standard practice in South Africa. These plantings make more efficient use of inputs such as land, fertilizer, irrigation and spraying, and they are capable of high early production and economic returns. However, disadvantages develop over time, depending on tree density and climate. These include overcrowding between and in rows, which induces difficult access by vehicles, poor insect control in dense canopies, and a decrease in yield and quality due to dieback and overcrowding. It is thus important to find a method to control tree size to maintain the benefits of such plantings. All three main tree size controlling methods viz. the use of genetic dwarf plant material, management practises and the use of transmissible agents are currently under investigation in the country. The aim of this study was to identify transmissible agents that can be used successfully to control tree size without detrimental effects. Research was done to identify transmissible agents as well as their application as dwarfing agents for commercial use. An important safety measure that should be considered before a transmissible agent is introduced for tree size control of citrus, is that such an agent should be endemic in the industry. Citrus tristeza virus (CTV) is a well known endemic disease in Southern Africa and virus-free shoot-tip grafted (STG) material is pre-immunised with avirulent isolates to reduce its effect. STG is also used to remove citrus viroids (CVd's) and the use of such agents as dwarfing agents is in contradiction with the aims of the Southern African Citrus Improvement Programme. However, research over many years in Australia has shown that avirulent CVd's can be used succesfully to control tree size. It is thus important to identify suitable CVd isolates for commercial application but it is also important to know which CVd's occur in commercial orchards. The occurrence of CVd's in old commercial citrus orchards, planted prior to the introduction of shoottip grafted material, was established by biological indexing with Etrog citron (Citrus medica L. cv. Arizona-861-S-1). Twenty-one percent of the trees indexed tested positive for CVd's. Sequential polyacrylamide electrophoresis (sPAGE) was employed to establish which viroids are most commonly present. Only two groups of CVd were detected, viz. citrus exocortis viroid (CEVd) and CVd-III. The latter CVd was the most common (40%) while CEVd occurred in only 8% of the samples. More than a third of the samples were infected with both CVd's. Two CVd isolates, a mild and a severe isolate according to Etrog citron reactions, were bud-inoculated to Delta Valencia on rough lemon, Poncirus trifoliata and four trifoliate hybrid rootstocks. Growth, production and occurrence of disease symptoms of these trees were compared to trees on the same rootstocks without CVd. All the trees were pre-immunized with the standard tristeza virus isolate. Results of sPAGE analysis of nucleic extracts indicated that the severe isolate (CD 11) contained CEVd only, and the mild isolate (CD 12) contained CVd-III. Overall, both isolates caused a significant reduction in tree size. The cumulative production over five years of the CD 11 infected trees did not differ from the CVd-free trees although the trees were smaller. This was due to a significantly higher production efficiency (kg/m(3) canopy). The production efficiency of the CD 12 trees was similar to the CVd-free trees, but the smaller trees resulted in a significantly lower production. Disease symptoms occurred with both isolates, but symptoms differed. Poncirus trifoliata var. Rubidoux was more sensitive to CVd isolates than four trifoliate hybrid rootstocks. Marsh grapefruit trees on Troyer citrange rootstock were bud-inoculated with different CTV isolates prior to planting in the field. Selected CTV isolates GFMS 2, GFMS 10, GFMS 12, GFMS 19, GFMS 25, GFMS 27 and GFMS 35, free of citrus viroids, were bud-inoculated into the virus-free plants. A severe isolate (GFSS 1) and plants that were left virus-free served as controls. Tree size, production, fruit size and tree health were determined. Fifteen years after planting, canopy volumes of trees with three isolates, GFMS 2, GFMS 19 and GFMS 25, were significantly smaller than the control trees as well as trees with isolates GFMS 10, GFMS 12 and GFMS 35. Trees with isolate GFMS 19 had a larger diameter than those with isolates GFMS 2 and GFMS 25. Together with a slightly higher yield efficiency, GFMS 19 trees resulted in a cumulative yield equal to that of the control and the GFMS 12 trees. Considering fruit size and their value, the performance of trees with isolate GFMS 19 equalled that of the larger trees. Tree health was also similar which makes this isolate suitable for use in high density plantings. A projection was made which showed that the production and crop value of to trees with isolate GFMS 19 were similar to those of trees with isolates GFMS 10, GFMS 12 and GFMS 35. However, benefits such as easier and better spray application and easier harvesting can increase profits when trees with isolate GFMS 19 are planted. The dwarfing characteristics of four isolates, CD 4, CD 8, CD 9 and CD 10, derived from healthy looking dwarfed field trees were evaluated. They were bud-inoculated to Delta Valencia trees on Yuma citrange rootstock prior to planting in the field. Five years after planting, isolates CD 4 and CD 9 successfully reduced canopy volumes by 60%, and CD 10 by 30%, without any detrimental effects. No CVd's could be detected biologically or by sPAGE in these three isolates. Isolate CD 8 however, contained two viroids, CEVd and CVd-III, but had no deleterious effects on the rough lemon rootstock. CTV was the only other pathogen present in the isolates. Indexing for cachexia, psorosis, impietratura and tatter leaf was negative. The dwarfing abilities of the isolates are therefore attributed to isolates of citrus tristeza virus. Production was according to tree size and the yield efficiency of the inoculated trees was equal to that of the uninoculated control trees. External and internal fruit quality was not affected. The trees were naturally infected with huanglongbing (greening) five years after planting, but the disease remained low for several years in trees with isolate CD 4. Three transmissible isolates (CD 4, CD 9, CD 10), derived from dwarfed field trees, were compared with two CVd isolates (CD 8, CD 12) for their abilities to control tree size of sweet orange. The isolates were bud-inoculated to Valencia on rough lemon, two Poncirus trifoliata and three trifoliate hybrid rootstocks, and compared to uninoculated trees on the same rootstocks. Isolates CD 4, CD 9 and CD 10 gave no reaction on Etrog citron and sPAGE of nucleic acid extracts failed to detect known CVd's. The two CVd isolates gave severe and mild reactions on Etrog citron and sPAGE showed that CD8 contained CEVd and CVd-III while CD 12 contained only CVd-III. The effect of the isolates on tree size, production, production efficiency and disease occurrence were monitored. Overall, CD 4, CD 9 and CD 10 did not reduce canopy volumes while trees with CD 8 and CD 12 were significantly smaller. However, CD 4 significantly reduced canopy volumes where Yuma citrange was used as a rootstock. In contrast, CD 8, containing CEVd, did not reduce canopy volumes on this rootstock, while CD 12 reduced it significantly. None of the trees on the other rootstocks were affected by either CD 4, CD 9 or CD 10. Canopy volume reductions by CD 8 and CD 12 differed from each other with all the trifoliate rootstocks. The production efficiency of trees with the two CVd isolates was significantly higher than the control trees as well as those with CD 4, CD 9 and CD 10. The higher efficiency of these trees resulted in cumulative production equal to the uninoculated trees. Disease symptoms occurred where all the isolates were inoculated, however, symptoms as well as the susceptibility of the rootstocks, differed among each other. Delta Valencia trees on Yuma citrange rootstock were inoculated respectively with two mild (GFMS 12 and T55), an intermediate (GFMS 10) and a severe (GFSS 1) CTV isolate prior to planting in the field. The same CTV isolates were also inoculated in combination with a CVd- III isolate. A virus-free control was included in the trial. All the CTV isolates were without the Seedling Yellows component of CTV. Seven years after planting, canopy volumes of the trees with the two mild isolates and the control were smaller than those of trees with the intermediate and severe isolates. This was in contradiction to what was expected. Overall, the CVd isolate had an additional reducing effect on canopy size, but only those trees with mild isolate T55 and severe isolate GFSS 1 were significantly affected. Production on a per tree basis was according to the canopy sizes, thus, the trees carrying the intermediate CTV isolate were the highest producers. The production efficiency (kg/m(3) canopy), however, did not differ among trees with the CTV isolates and the control. The CVd isolate generally increased the production efficiency. The internal quality of the fruit was not affected by any treatment. The lack of a suitable genetic dwarfing rootstock for citrus makes it essential to evaluate alternative methods to reduce tree size for high density plantings. Four transmissible dwarfing factors, derived from dwarfed trees, were evaluated for commercial application in a hot (Malelane) and intermediate (Nelspruit) production area. Generally, trees in the hot production area were more vigorous with a lower production efficiency than trees in the intermediate area. The dwarfing effects of isolates CD 4, CD 9 and to a lesser extent, CD 10, were reduced in the hot area. Isolate CD 8 caused no dwarfing at either location. The reduction of dwarfing at the hot site may be attributed to the suppression of CTV by high temperatures. Currently some of the isolates which were tested in this investigation are applied on a larger scale in different climatic areas as commercial trials. Formal trials are continuing and they are aimed to elucidate the dwarfing characteristics as well as the inducement of disease of the four viroids in the CVd-III group.
The characterisation of turkey rhinotracheitis virus from chickens and the development of a suitable vaccine.
(1994) Maharaj, Sanjay Balkishore.; Da Graca, John Vincent.
Three turkey rhinotracheitis virus-like (TRTV-like) isolates were isolated from chickens with swollen heads. All grew well via the yolk sac (y/s) , chorioallantoic membrane (CAM), tracheal organ culture (TOC) , chicken embryo fibroblast (CEF) , and Vero cell routes. Affected embryos were stunted and severely congested. No pathological alterations were detected in allantoic sac (a/s) inoculated embryos. The CEF and Vero cells required trypsinisation for five consecutive passages before any visible cytopathic effects (CPE) were observed. Intra-cytoplasmic eosinophilic inclusions were observed in Vero and CEF monolayers . Only isolate 652/93 caused 100% ciliostasis in TOC. The other two isolates were able to cause a maximum of only 70-80% ciliostasis. The isolates were inactivated by chloroform treatment and exposure to 56°C for 1 h. Long term storage could be achieved at -70°C or in liquid nitrogen but not at 4°C or at -20°C. The isolates did not agglutinate chicken red blood cells and were found to contain genomes of RNA. They were able to elicit TRTV antibodies in specific pathogen free (SPF) birds as measured with the Pathasure enzyme linked immunosorbent assay (ELISA) kit. They could also be neutralised by TRTV-specific antisera. Electron microscopy of infective allantoic fluid (A/F) revealed particles of 100-300 nm in diameter with a helical nucleocapsid component approximately 15 nm in diameter and a fringe of approximately 12 nm long spikes. The processes of VLP development and maturation in TOC's and Vero cells were similar with accumulations of virus-like nucleoprotein close to the plasma membrane, forming the nucleocapsid. Virus-specified spikes were then inserted into the plasma membrane after which the VLP budded from the plasma membrane, incorporating this membrane with spikes as its own. Nine viral polypeptides with molecular weights of 200kDa, 83kDa, 53kDa, 40kDa, 37kDa, 28kDa, 19kDa and 15kDa were detected by SDS-PAGE in samples of the three isolates. The 83kDa and 53kDa polypeptides were also detected by western blotting using TRTV specific antisera. Both, a TRTV and a 652/93 isolate non-radioactive DNA probe, appeared specific for TRTV and TRTV-like isolates. The 652/93 probe detected 652/93 virus in SPF chickens for 19 days post-inoculation. A vaccine produced in SPF eggs using the attenuated 652/93 isolate, was able to protect vaccinates against virulent virus in laboratory challenge studies. In field trials, birds vaccinated at day-old or at day-old and again at 14 days, showed slightly improved performance compared to non-vaccinated birds. However, this benefit was not statistically significant. It is suggested that other environmental and disease factors mask the benefit provided by the vaccine in field trials. The three TRTV-like isolates appear to be chicken strains of TRTV and vaccination with an autogenous vaccine appears to afford some benefit to vaccinates.
A study on avocado sunblotch disease.
(1980) Da Graca, John Vincent.; Martin, Michael Menne.
Avocado sunblotch disease is a graft-transmissible disorder known for over 60 years and has now been recorded in at least eight countries around the world. Affected trees develop yellow, depressed streaks on young stems and fruit, marked rectangular cracking of the mature bark and a decumbent style of growth. Often a tree with symptoms produces completely symptomless shoots, termed 'recovery' growth, which are latently infected. There is a reported 95 to 100% transmission of sunblotch through the seed of such branches, and "the resultant seedlings are themselves symptomless. Indexing for sunblotch to ensure that scion and, in view of seed transmission, especially rootstock material is free of the disease is very important . The standard method used for many years has been to graft tissue onto healthy indicator seedlings and observe for symptom development for 18 months to two years. One aim of the study presented in this thesis was to develop more rapid methods for detecting the sunblotch agent. By conducting the standard indexing method in a glasshouse at controlled high temperatures of 30/28º C (day/ night) and by cutting back the indicator plants every three months, the time was reduced from two years to eight months. While this represents a considerable saving in time, the ideal must be to develop a laboratory diagnostic test that requires no more than a few days, at most, to complete. A comparative study was therefore initiated on the phenol metabolism of healthy and sunblotch-infected avocados to determine whether infection causes any major change that may reliably serve as a marker for diagnostic purposes. Significantly increased peroxidase (PO) and phenylalanine ammonia-lyase (PAL) activities, decreased indoleacetic acid (IAA) oxidase activity and higher sinapic acid levels were detected in bark tissue showing sunblotch symptoms, but not in symptomless 'recovery' growth. In contrast, increased polyphenoloxidase (PPO) activity and isoenzymes, total soluble protein levels, water soluble phenols and reduced ferulic acid levels were found in the bark of all infected trees tested, both with symptoms and symptomless. However, these latter changes have been associated with other plant-virus systems and are therefore not necessarily specific for sunblotch. Neither is any sufficiently large to be definitive as a diagnostic test. Two unidentified phenols were detected in infected, mature bark, but not in infected young bark and leaves. introduced the possibility of rapid disease detection by polyacrylamide gel electrophoresis (PAGE) of extracted RNA's as used for known viroids. In this study the presence of previously reported small molecular weight sunblotchassociated RNA's was confirmed using PAGE methods requiring two to four days to complete. This thesis presents as a further development a more rapid method of PAGE detection of RNA's enabling indexing for sunblotch to be completed in under six hours. Whilst the biochemical studies did not reveal diagnostically meaningful differences between healthy and infected avocados, there were tendencies towards differences between healthy and symptomless carrier tissues, further investigation of which may lead to a future understanding of symptom development and the symptomless condition. These include apparent higher PO and lower PAL activities in symptomless carrier tissue, as well as higher PO isoenzyme a[1] and lower IAA oxidase isoenzyme a[1] activities. General studies on sunblotch-infected avocados showed that fruit from symptomless 'recovery' growth branches are significantly larger and have a higher oil content than those from healthy or diseased branches, the latter finding possibly indicating a more advanced state of maturity of 'recovery' growth fruit due to earlier flowering. The avocado sunblotch agent was shown to have an in vivo thermal inactivation point of 55º C, a temperature higher than the avocado tissue can withstand thereby eliminating the possibility of thermotherapy of infected twigs. In a host range study four lauraceous plant species, Persea Schiedeana, Cinnamomum zeylanicum, C. camphora and Ocotea bullata, were successfully infected with sunblotch by grafting from infected avocado. This is the first demonstration of any host other than avocado. A phanerogametic member of the same family, Cassytha filiformis, was shown to be able to transmit the disease from avocado to avocado. No hosts from other families were found. During an electron microscope study of sunblotch-infected avocado leaf tissue, gross alterations of the chloroplasts in the yellow areas were observed. These changes included organelle swelling, loss of grana and stroma lamellae, rearrangement of remaining membranes and presence of vesicles. Also in the yellow areas paramural bodies were encountered in higher numbers and displaying altered structure than in healthy and symptomless infected leaf tissue. This study on avocado sunblotch disease was successful in both of its aims. Firstly with regard to quicker indexing techniques, the standard method using indicator plants was shortened from two years to eight months, while a rapid, six-hour test based on PAGE analysis, was developed. Secondly, more light has been shed on the biochemical and ultrastructural effects of sunblotch on its host, the avocado, as well as providing information regarding the thermal sensitivity and the host range of the agent.

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Browsing Doctoral Degrees (Plant Pathology) by Author "Da Graca, John Vincent."