Plant Pathology

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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.
An epidemiological analysis of the Phytophthora and Alternaria blight pathosystem in the Natal Midlands.
(1980) Putter, Christoffel Antonie Johannes.; Martin, Michael Menne.; Rijkenberg, Fredericus Hermanus Johannes.
The history of the development in Natal of a forecasting service to warn of outbreaks of late blight disease caused by Phytophthora infestans is presented. The late blight pathogen and Alternaria solani, the causal organism of early blight disease, interact on potatoes and tomatoes to form a blight disease complex. Evidence is presented to show that it is expedient to manage this blight complex as a whole rather than to direct control at only one of the components in ignorance of the consequential enhancement of the potential of the other. In a search for an improved blight complex management strategy, factors concerning the possible existence of an annual migration of Phytophthora infestans inoculum, first postulated in the 1960's, along an east-west route across Natal, are collected and collated. Corroboration of the existence of the Phytophthora-pathway is given, inasmuch as it represents a serial outbreak of late blight along a temporal gradient. The possibility that the pathway is a manifestation of disease resulting from the erruption of pre-existing inoculum along an environmental gradient, can not specifically be excluded. However, the peculiar pattern of anabatic and katabatic winds along a river-valley network, superimposed on a continuous cropping pattern and its concomitant opportunity for blight to be endemic in the province, supports the postulated Phytophthora-inoculum pathway A fungicide spray trial was conducted in order to investigate the possibility of us i ng the pathway phenomenon as the framework for an improved blight control strategy and to explore the nature and level of the competitive interaction between Phytophthora infestans and Alternaria solani. This trial revealed that the interaction between the components of the blight complex was differentially altered by weather patterns and fungicide combinations. Treatments in which metalaxyl (Ridomil) alone was used for the control of late blight, gave a yield similar to those with propineb (Antracol), which inhibits A. solani primarily but also hus some negative effect on P. infestans. The yields from both these treatments were siguificant ly (p < 0,05) better than the yields recorded in the unsprayed control plots. A treatment in which Ridomil and Antracol were combined such that each was applied according to its recommended concentration, gave yield increases of 32,3% over the unsprayed control, although the yield from the Ridomil/Antracol treatment was not significantly greater (p < 0,05) than the yields recorded where either Ridomil or Antracol were used. A computer simulator, named GAUSE, was developed to simUlate the consequences of the competition between various combinations of P. infestans and A. solani. Results simulated by GAUSE corroborated those obtained from the field trial and support the conclusion that diseases of complex etiology require more than simplistic, univariate analysis of single cause-and-effect pathways. The competition quotient CQ is developed as a new parameter of competitive interactions. It is calculated as the ratio of the amount of disease in the absence of competition, to the amount of disease when the causal pathogen is competing with another pathogen in the same niche. The CQ may be calculated from various standard epidemiology statistics and it is used to demonstrate that the competitive displacement phenomenon places constraints on the interpretation and application of Vanderplank's basic epidemiology equations. A new pathosystems management concept namely the pathotope (pathos = suffering; topos = place0 concept, is introduced, having developed from the notion that epidemics have spatial as well as temporal attributes. Accordingly, an area in which individual farms are at the same level of probability at risk to disease, delimits the pathotope. The concept can be described at many integration lsvels and is presented as an important quantitative unit of comparative epidemiology. The pathotope concept accomodates such notions as are contained in the postulated Phytopnthora-pathway and is especially suited to integration with disease forecasting methods. An example of the application of the pathotope approach is presented and a strategy is proposed by which fungicide spraying is initiated and applied synchronously as determined by the degree of communal risk to attack and epidemic increase of disease. Within a pathotope, several common factors collectively determine the vulnerability of the group to disease. If a coherent, uniform strategy is to be developed and implemented by pathotope members, it is necessary that all members have access to the relevant information and that it be collected and disseminated conveniently and rapidly. A computer-based disease monitoring and mapping system which achieves these objectives is presented.
Studies on the pathogenicity of Puccinia graminis f. sp. Tritici and the nature of host resistance in wheat.
(1986) Le Roux, Jacobus.; Rijkenberg, Fredericus Hermanus Johannes.
This study was undertaken to investigate the pathogenic variation of Puccinia graminis f.sp. tritici in South Africa and to determine the nature of host resistance to this fungus. Seventeen pathotypes of wheat stem rust were identified in 1981 and only one during 1982 and 1983. The 1984 and 1985 surveys provided six and nine pathotypes respectively. These included two new pathotypes 2SA100 and 2SA101, both virulent for Sr24. No avirulence was detected for wheat stocks having Sr7a, 8a,10,11,14,16,18,19,20 and 28. No virulence was found on wheats with Sr8b,13,21,22,25,26,27,29,31,32,35,dp2 and Tt3. Data suggested that the pathogenicity pattern in South Africa is strongly influenced by the resistance genes present in wheat cultivars. Pathogenic studies of pathotypes 2SA100 and 2SA101 cultivars demonstrated that both possessed increased virulence for Sr24 and appeared to be mutations of earlier types. Seedling and adult plant response studies indicated genetic vulnerability in 60% of the locally cultivated cultivars. Nine of the 23 recommended cultivars possess Sr24, either alone or in combination with other resistance factors. Pathotypes 2SA100 and 2SA101, or races similar to these, constitute a major threat to wheat production in Southern Africa since they combine increased virulence with aggressiveness and good survival ability. Genetic studies showed that resistance was conferred by one dominant gene in each of Belinda (Sr5), SST33 (Sr9e), SST66 (Sr9e), SST102(Sr24), Gamka (Sr24), Gamtoos (Sr31), Karee and Betta. Two partially dominant genes were identified in Wilge, Gouritz (Sr36+) and SST23 (Sr24+). Resistance in Palmiet was conferred by one dominant, (Sr24) and one recessive gene, whereas one recessive and one dominant gene operated in Molen. Tugela carried three partially dominant genes, one of which was positively identified as Sr5. Palala also has three unidentified dominant genes. The study determined that inheritance of resistance in 15 South African cultivars was relatively simple with little genetic diversity. Three spring wheat cultivars, SST44, Palmiet and Gamtoos were used to study the quantitative aspects of specific resistance to P. graminis tritici. Averaged over three cultivars, latent period was extended by 57% and uredinium density was reduced by 29% relative to the susceptible check Morocco. Latent period appeared to be a race-nonspecific resistant component of all three cultivars. This study indicated that the variation in uredinium density associated with specific resistance was similar to that reported by other workers for compatible host pathogen interactions.
Studies on Puccinia recondita f. sp. tritici with special emphasis on adult plant resistance in wheat.
(1986) Pretorius, Zacharias Andries.; Rijkenberg, Fredericus Hermanus Johannes.; Wilcoxson, Roy Dell.
Leaf rust (Puccinia recondita f.sp. tritici) of wheat (Triticum aestivum) was widespread in South Africa during 1983, 1984 and 1985 and often reached epidemic levels, especially on autumn-sown spring wheat in the Cape Province. Nine physiologic races were identified during the study period. The most common race was avirulent to the leaf rust differential genes Lr3a, 3bg, 3ka, 11, 16, 20 and 30 and virulent to Lr1, 2a, 2b, 10, 14a, 15, 17, 24. Resistance genes Lr9, Lr19, Lr21 and Lr26 were effective to all isolates tested. Evaluation of wheat genotypes for components of resistance, viz. infection type, latent period, number of uredinia and uredinium size, revealed three phenotypic reaction classes. The first group exhibited negligible resistance, the second was susceptible or moderately susceptible as seedlings but resistant as adult plants while the third group was resistant at all growth stages tested. Adult plant resistance was expressed by hypersensitive or non-hypersensitive reactions and the combination of components conditioning resistance varied. Adult plant resistance conferred by gene Lr22a was characterized by a long latent period, small uredinia, reduced sporulation and an absence of a differential interaction between components of resistance and different races of Puccinia recondita f.sp. tritici. Numbers of uredinia on flag leaves of RL6044 (Lr22a) were equal to those of a susceptible check, Line E. Lr22a was inherited as a partially recessive gene in crosses with Zaragoza and SST33. Assessment of latent period, number of uredinia and infection type in F4 and FS families homozygous for Lr22a and derived from crosses between RL6044 and Zaragoza or SST33, revealed significantly different levels of resistance between families. Differences were attributed to other genes modifying the expression of Lr22a. Adult plant resistance of Era, Glenlea, RL6044 and sinton was expressed prior to the fifth-leaf stage. Latent period increased and number of uredinia decreased as each wheat matured. While the latent period of the flag, flag-l and flag-2 leaf was similar within Era, Glenlea and RL6044, differences between these genotypes occurred. The latent period of flag leaves of Sinton was shorter than that of the two lower leaves. Significantly fewer uredinia developed on the flag-2 leaf of Glenlea. A reduction in temperature from 21 C to 15 C significantly increased latent period in Era, Glenlea and RL6044, and also restricted uredinium size on flag leaves of RL6044. The adult plant resistance of Glenlea crossed with Line E was conferred by two partially recessive genes. Additionally, F2 to FS progenies of this cross eXhibited high levels of hypersensitive seedling resistance at 29 - 31 C to certain isolates. The latter resistance was not conferred by Lr1 or by the LrT2 gene for mature plant resistance in Glenlea. The high-temperature expression of resistance could be due to a second gene for adult plant resistance or to a previously undetected seedling gene.
A study on the ultrastructure and control of the gladiolus rust pathogen, Uromyces transversalis.
(1988) Ferreira, Johan Francois.; Rijkenberg, Fredericus Hermanus Johannes.
The substomatal vesicle of Uromyces transversalis develops in five distinct stages after the formation of a single infection peg in the host plant Gladiolus spp. The primary hyphae of the substomatal vesicle orientate at right angles (rather than at acute angles or parallel) to the stomatal slit and the parallel veins of a gladiolus leaf or to those of the non-host, Zea mays. The transverse orientation of Uromyces transversalis uredia is apparently a genetically controlled phenomenon. The two primary hyphae normally develop asynchronously on opposite sides of the substomatal vesicle, and, after the formation of a haustorium mother cell, secondary hyphae branch off. These subsequently form basal cells each of which give rise to one or more holoblastic protruberances on its distal surface. A septum delimits the protruberance from the basal cell to form a urediospore initial, which in tum elongates and, by septum formation, forms a pedicel and a urediospore. The urediospore is seceded mechanically through the process of schizolysis. The pedicel of a spore thus formed remains on the basal cell and becomes a collar around the next protruberance. The basal layer of the two-layered septum, that delimited the pedicel from the basal cell, grows out to form the wall of the subsequent protruberance, in the process rupturing and laterally displacing the terminal septal layer. A new basipetal septum forms to delimit the subsequent urediospore initial. Therefore, successive urediospores are formed enteroblastically and give rise to a series of basipetal collars. Cultivars, naturally occurring species and breeding lines of gladiolus were evaluated for rust resistance. The cultivars showed no sign of resistance, whereas the populations of wild species and breeding lines had similar resistance profiles. The species G. daleni and G. tristis var. tristis showed promise for breeding. Infection of these species caused an almost immune, fleck reaction. The resistance of the species G. daleni was manifested by the abortion of primary hyphae prior to the formation of the haustorial mother cell. The intercellular hyphae of gladiolus rust only partially adhered to the mesophyll cell of the resistant host. The haustorial apparatus that did form in the mesophyll cells in the resistance reaction was inhibited at various stages of its development. The rust disease could be controlled chemically by either bitertanol or triadimefon. Triadimefon, however, caused a shortening of the internodes of the flower spike. The early development of U. transversalis infection structures in bitertanol-treated leaves was inhibited at, or shortly after, substomatal vesicle development. A number of mature substomatal vesicles with intercellular hyphae, however, did develop in the bitertanol-treated leaf tissue, probably because this fungicide undergoes limited translocation in the gladiolus lamina.
Studies on the expression of resistance of Coffea selections to Hemileia Vastatrix.
(1990) Coutinho, Teresa Ann.; Rijkenberg, Fredericus Hermanus Johannes.
Physiological races of Hemileia vastatrix in southern Africa were identified. Prevalent races were I (2.2%), II (88.9%) and III (2.2%). Six samples could not be identified. Twelve biotypes of race II were distinguished. In some cases, the biotypes only occurred in specific regions. It was established, using fluorescence microscopy, that, in some cases, the percentages of germinated urediospores that did not form appressoria, appressorium formation over stomata, and aborted appressoria, were significantly different between susceptible and resistant selections of the host, and non-hosts. The sequence of events leading to successful infection was investigated using scanning electron microscopy (SEM). When a stoma is encountered by a germ tube tip a uniquely shaped appressorium forms over one end of the stomatal slit. A distinct appressorial foot is wedged within the stomatal vestibule. In coffee, a torpedo-shaped substomatal vesicle initial (SSVI) develops bilaterally from the apex of the infection wedge, While in bean, the infection wedge protrudes into the substomatal chamber. The substomatal vesicle (SSV), at 48 hours post inoculation (hpi) is anchor-shaped. Haustorial mother cells are formed on stubby primary infection hyphae which curve back onto subsidiary cells. No differences in appearance of these structures were noted between resistant and susceptible coffee selections. A much-branched mycelium ramifies through the intercellular spaces of the mesophyll cells 96hpi. In bean, the SSV began to collapse 48hpi. Bayfidan ® only slightly suppressed fungal development on the leaf surface. However, within susceptible tissue, this systemic fungicide had an effect on the morphology of the fungus. Extracellular material accumulated on the SSVI and the SSV. The SSV appeared swollen, and disruptions in the vesicle wall was noted. The discovery of teliospores on locally infected trees led to a SEM study on their structure, development and germination. Infection structure formation on the leaf surface, latent period, reaction score and urediosorus concentration differed between susceptible coffee leaves of different ages. Generally, mature leaves are more susceptible than very young or old leaves. A range of fungicides, mainly systemics, were tested in the field on naturally infected coffee trees. Various epidemiological and climatic factors influence rust development in the field. The role of these factors at the fungicide site and in commercial coffee-growing regions of southern Africa was evaluated.
Studies on the resistance of wheat and maize to fungal pathogenesis.
(1990) Van Asch, Michiel Alfred Jan.; Rijkenberg, Fredericus Hermanus Johannes.
17-Day-old seedlings of winter-wheat cultivar SST25 were inoculated with an avirulent race of wheat leaf rust, Puccinia recondita f.sp. tritici. After various time intervals the plants were reinoculated with a virulent wheat leaf rust race. No change in latent period or infection type was observed. However, the infection frequency was reduced by approximately 60 per cent. The phytotoxic effects of three mycotoxins of Fusarium spp. (fumonisin B(1)(FB)1) , moniliformin and T-2 toxin), and pathotoxin extracts of Exserohilum turcicum (HT-toxin) and Stenocarpella macrospora (SM-toxin) were studied using callus from the scutella of immature cobs of maize, Zea mays. The callus was grown on modified MS medium containing either 0, 0.1, 1.0, 10, or 100 mg (or ml) toxin per litre. For SM-toxin the concentrations used were 0, 0.01, 0.1, 1.0 or, 10 ml/l. Mass increase of callus on medium containing FB 1, moniliformin, T-2 toxin, and HT-toxin decreased as the concentration of toxin increased, resulting in a significant reduction at the highest toxin level. SM-toxin callsed a slight reduction in mass at 0.01 ml/l, but stimulated growth at 1.0 ml/l. At 10 ml/l a significantly lower callus mass increase was recorded. Transmission electron microscopy studies of FB 1-treated callus showed an increased level of activity in the toxin-treated cells resulting in thicker cell walls, occurrence of starch grains and phenolic substances, when compared to the control. The mitochondria of callus cells were affected by SM-toxin, and starch was found in all toxin treatments. When transferred to toxin-free medium after treatment with FB 1, a complete recovery of the callus occurred at all toxin levels but the highest, although regrowth occurred at this level. Callus treated with SM-toxin retained the same growth rate as during the toxin treatment, and it can be concluded that the toxin has a permanent effect on the growth rate of callus. Maize seedling leaves, injected with a 10g/l FB(1)-solution at the stalk base, showed necrotic areas and chlorotic flecks. The toxin-treated plants were stunted and occasionally produced side shoots. S. macrospora-susceptible and -resistant seedlings, injected in a similar fashion with SM-toxin, gave a different response to the toxin. Susceptible plants were affected by the toxin, while no effects were observed in resistant plants.
Studies on the expression of resistance to stem rust of wheat caused by Puccinia graminis f.sp. tritici.
(1991) Lennox, Cheryl Lynne.; Van Staden, Johannes.; Rijkenberg, Fredericus Hermanus Johannes.
The endogenous cytokinin levels of healthy primary leaves and seeds of a stem-rust susceptible wheat cultivar Little Club were compared with those of Little Club containing the stem rust resistance gene Sr25. Use was made of paper, column and high performance liquid chromatography techniques to separate the endogenous cytokinins in the plant material, and the soybean callus bioassay was used to test for cytokinin-like activity of the chromatography fractions. Leaf material of the resistant Little Club Sr25 had a higher level of total cytokinin activity than Little Club, whereas seed material of Little Club Sr25 did not always have higher levels of cytokinins than Little Club. A number of cultivars would have to be tested before the usefulness of cytokinin levels as an indicator of resistance could be determined. The development of urediospore-derived infection structures of Puccinia graminis f.sp. tritici in wheat, barley, sorghum and maize was examined by scanning electron microscopy (SEM). Infection on and in the four species followed a similar pattern up to, and including, primary infection hyphae formation. In wheat, barley and maize, when a primary infection hypha abutted onto a host epidermal cell, a septum was laid down delimiting a primary haustorial mother cell (HMC); primary HMCs did not form in sorghum. Secondary infection hyphae arose on the substomatal vesicle side of the primary HMC septum; infection did not progress further in maize, but in wheat and barley secondary infection hyphae branched, and proliferated intercellularly forming the fungal thallus. Secondary HMCs were delimited when an intercellular hypha abutted onto host cells. In all four species atypical infection structures were also observed. In an attempt to determine the timing and expression of stem rust resistance gene Sr5, infection structure development of Puccinia graminis f.sp. tritici race 2SA2 in a resistant line (ISr5Ra) and a susceptible line (ISr8Ra) was compared quantitatively using a fluorescence microscopy technique. The results indicated that there were no significant differences in numbers of specific infection structures observed in the two near-isogenic lines up to, and including, 48 hpi, by which time race 2SA2 had successfully formed secondary HMCs in both lines.
Studies on Alternaria porri and Stemphylium vesicarium on Allium spp.
(1993) Aveling, Theresa Ann Sheila.; Rijkenberg, Fredericus Hermanus Johannes.; Wehner, F. C.
During surveys in South Africa, Alternaria porri (Ellis) cif. and Stemphylium vesicarium (Wallr.) E. Simmons were found to be destructive seed-borne pathogens of onion (Allium cepa L. ). These two pathogens are also reported on garlic (Allium sativum L.) in South Africa for the first time. The development and morphology of conidiophores and conidia of the two pathogens on the onion leaf surface were examined using scanning electron microscopy. In both pathogens, solitary or fasciculate conidiophores emerged through the epidermis. Bud-like conidial initials were produced singly at the apex of conidiophores. As conidia of S. vesicarium matured, they became oblong to ovoid and densely verrucose. Those of A. porri showed slight growth in width but pronounced elongation. Conidial germination, formation of pre-penetration structures, penetration of the onion leaf surface by A. porri and S. vesicarium, and the subsequent infection process by A. porri, were studied using light, scanning electron and transmission electron microscopy. Conidia of both pathogens usually germinated within 24 h of inoculation, forming several germ-tubes which often terminated in bulbous appressoria produced directly on the epidermal cells or on stomata. Following direct penetration of the outer epidermal cell wall or the stoma, bulbous primary hyphae developed below the appressoria. Secondary hyphae of A. porri developed from primary hyphae and grew within the intercellular spaces, penetrating mesophyll cells. The changes in ultrastructure of infected cells, and of cells in close proximity to secondary hyphae, are described. six fungicides, anilazine, benomyl, carbendazim/flusilazol mixture, procymidone, tebuconazole and thiram, as well as a hotwater soak (50 C for 20 min) and sodium hypochlorite treatment, were evaluated for their efficacy in reducing both pathogens on seed and in culture. The effect of the various treatments on seed germination, and seedling emergence and growth, was determined. None of the treatments eradicated A. porri and S. vesicarium from onion seeds. The hot-water soak proved to be the best treatment for reducing these pathogens, although percentage germination and emergence of onion seeds were reduced when compared to the control.
The use of Agrobacterium for plant improvement.
(1994) MacRae, Sharmane.; Van Staden, Johannes.; Thomson, Jennifer A.
Agrobacterium species have potential as tools for plant improvement, in that they can be used as biological control agents against crown gall disease, vectors for gene insertion into plants and plant root-inducing bacteria. For a bacterium to be successful as a biocontrol agent against crown gall disease, it must be able to produce an effective agrocin specific for A. tumefaciens pathogens and be able to colonise host plants effectively. Successful biological control of crown gall disease has been achieved on a limited range of host plants by application of Agrobacterium radiobacter. Strain K84 is non-pathogenic, produces an antibiotic type substance, agrocin 84, which kills a specific spectrum of pathogenic Agrobacterium strains, and is a good plant coloniser. The colonisation abilities of this bacterium and a number of potential biocontrol bacteria, D286, 173 and its derivatives, H8, and H6 and its derivatives, were compared in vitro and in vivo. In addition the ability of these strains to control crown gall pathogens in vitro and in vivo was assessed. Although D286 and H8 were good long term colonisers they were subsequently eliminated from the programme because D286 was unable to control grapevine crown gall disease, and H8 was a pathogen, with a narrow host range for biotype 3 pathogens, which had not been cured. Good long term colonisation ability was shown to be critical to the success of the biological control strain. Both K84 and J73 strains produced fibrils which attached them to tomato root surfaces and have similar colonisation efficiencies up to 14 days after inoculation. However, the ability of J73 to colonise plants for longer periods was significantly less than that of K84. Thus, the presence of fibrils is not sufficient to ensure colonisation. No correlation was found between hydrophobicity and colonisation. Poor in vivo biological control of tomatoes and grapevines by J73 and its derivatives has been shown to be due to poor long term colonisation. The additions of the agrocin 84 plasmid to J73 improved in vitro biological control by J73 but was not effective in vivo. Thus although J73 produced a broad host range agrocin it was ineffective as a biocontrol agent due to its poor long term colonisation ability. H6 and its derivative proved to be good long term colonisers of the grape vine rhizosphere and were able to effectively control crown gall disease on grapevines. Eucalyptus grandis was propagated in vitro from axillary buds. The effect of the gelling agents gelrite, agarose and agar on propagation was determined. Shoot multiplication and elongation on gelrite-containing media was found to be superior to that obtained on agarose- and agar-containing media. Rooting was enhanced with gelrite as the gelling agent. In vitro clone development from seed of selected E. grandis genotypes and the integration of this into vegetative clonal propagation programmes is proposed. Not all clones developed in this manner rooted well under in vitro conditions. Thus the potential use of A. rhizogenes to improve rooting was investigated. A. rhizogenes is a bacterial pathogen which causes 'hairy root' disease on certain plants. Eucalyptus species do not fall into the natural host range of this organism, however, it is able to infect these plant species. In an attempt to improve rooting and root quality of in vitro and in vivo propagated Eucalyptus species and clones, the root-inducing genes on the Ri plasmids of a number of A. rhizogenes strains were inserted into Eucalyptus by inoculating in vitro and in vivo stem cuttings with the selected A. rhizogenes strains. The resultant chimeric plants have transformed roots and normal shoots. Root development was monitored in vitro and after the plantlets had hardened off, and in vivo. Only transformed roots grew as root cultures in hormone-free liquid medium. The potential use of this procedure for improving rooting of clonal material is discussed. Under in vitro conditions for example, one of the broad host range A. rhizogenes strains, LBA9402, was able to induce up to 80 % rooting on E. grandis, E. nitens and E. dunnii explants. While under nursery conditions for example, one of the two E. globulus clones tested, HM15, developed up to ten times as many roots in response to two strains of the A. rhizogenes bacterium (LBA9402 and TR8,3) while the other clone failed to respond. Not only the inherent rooting abilities of the numerous Eucalyptus genotypes and clones tested, but also hormone induced-rooting and Agrobacterium-mediated rooting were found to vary from clone to clone and genotype to genotype. A. rhizogenes strains were however, able to overcome the genetic control on rooting of certain clones but this was found to be dependent on both the genotype of the bacterium and the plant. The novel concept of using bacterial cocktails to improve rooting was investigated, with the aim of overcoming the limited host range of individual A. rhizogenes strains. Both in vitro and nursery trials were established to test the potential use of bacterial cocktails as a means of improving rooting of a range of Eucalyptus genotypes and clones. Agrobacterium cocktails not only improved the ability of certain clones and genotypes to root but also the quality of the roots. The anatomy of transformed roots from chimeric plants was compared to that of non-transformed roots, revealing no difference in root anatomy of these roots. Nutrient uptake studies using a radioisotope nutrient uptake bioassay showed no difference in phosphorus, potassium and nitrogen uptake by transformed and nontransformed roots. The potential of A. rhizogenes strains to improve rooting of two other species, Anacardum occidentale (cashews) and Pinus (a number of pine hybrids) was also shown in preliminary trials.
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.
Maize root rot in South Africa.
(1995) Hugo, Elbé.; Rijkenberg, Fredericus Hermanus Johannes.
Numerous fungal species are known to infect maize roots and eventually cause rot. The spectrum of fungi differs over localities as well as their relative frequencies. Fungi isolated from discoloured root tissue and root tissue without visible discolouration were classified as root pathogens and root colonizers, based on their isolation frequency from the respective samples. Exserohilum pedicellatum, Macrophomina phaseolina and Fusarium oxysporum were classified as the major root pathogens and Phoma spp., Curvularia spp. and F. chlamydosporum as root colonizers. Fungi classified as root pathogens tended to occur early in the growing season in juvenile tissue as opposed to root colonizers which occurred later. Treatments in an existing long-term water stress trial included no stress (irrigation), normal rainfall, stress until flowering, stress after flowering until mid grain-fill, stress after flowering and total stress. Root pathogens were isolated at higher frequencies in the no stress and normal rainfall treatments than in the total stress treatment. A positive linear relationship between the water stress index and the isolation frequency of M. phaseolina was obtained. Negative, non-linear relationships were, however, recorded for E. pedicellatum, F. oxysporum and F. moniliforme. The effect of tillage practices on disease severity was carried out in field trials at two localities. Tillage practices applied included rip, plough, chisel plough and no-till treatments. Significant differences between isolation frequencies and tillage treatments were recorded for F. oxysporum at Bloekomspruit and Trichoderma spp., Alternaria spp. and M. phaseolina at Mmabatho. Differences in isolation frequency of fungi involved in maize root rot, were determined in a crop rotation system where maize was rotated with soybeans, sunflowers and groundnuts. Crop rotation had a significant effect on the isolation frequency of Thielavia ferricola, E. pedicellatum, F. moniliforme and F. graminearum. The effect of crop rotation, however, seems to be complex since fungi were affected differently in the various treatments.
The epidemiology and control of Capsicum viruses in Natal.
(1995) Budnik, Krzysztof.; Da Graca, John Vincent.; Laing, Mark Delmege.
Virus diseases pose a serious threat to commercial pepper (Capsicum annuum L.) production in Natal. Following a survey of the principal capsicum-producing areas, potato virus Y (PVY) was found to be the predominant virus infecting peppers, often reaching 100% incidence. Currently, TSWV incidence and CMV levels are insignificant with respect to pepper crops in Natal. Thus, the diverse ecological and epidemiological factors which determine PVY infection of peppers were investigated. The potential host range of PVY was established in a glasshouse study. Seed from solanaceous weeds commonly occurring in vegetable-producing areas of Natal was collected. Seedlings were mechanically inoculated with a pepper strain of PVY and assayed for infection using double-antibody sandwich ELISA. Nicandra physaloides L., Solanum elaegnifolium Cav., S. nigrum L., S. velosum L. and S. aculeastrum L. were found to be susceptible to PVY infection. In addition, a field survey of over 100 samples of commonly occurring weed species growing in or adjacent to capsicum crops in the Pietermaritzburg and South Coast regions of Natal was carried out. Several weed species were found to be naturally infected with PVY, including Acanthospermum hispidum DC., Bidens pilosa L., N physaloides and S. nigrum. The spread of PVY into a pepper crop on the Natal South Coast was monitored during 1993. Virus spread was rapid, with PVY first detected in pepper seedling one week after planting, suggesting a nearby source of the virus. A survey of the wild vegetation prior to planting of the crop, revealed that populations of N physaloides may be the primary sources of PVY infecting the crop. Large virus-infected S. nigrum populations appeared later in the season, suggesting its role in maintaining high levels of PVY during periods when no pepper cultivation takes place. In addition to identifying possible virus reservoirs, several virus control measures were investigated, demonstrating ways of avoiding or minimising infection. The effects of insecticides, oil sprays (Virol), insect repellents (AzatinTM), yellow polyethylene traps and plastic mulches on virus incidence within peppers were evaluated in field experiments. Results of weekly sprays of the insecticide mercaptothion at 5%, increased virus incidence in peppers by 15% when compared to the untreated control. Similarly, the effects of insecticide applications on pepper yields and quality were negative. Results of applications of Virol at 1 % and Azatin™ at 1.5% did not differ from those of the unsprayed control. Mulching was most effective by reducing virus incidence in treated plots by 50% and resulted in a yield increase of 62% and a 40% increase in fruit quality. The use of yellow sticky traps reduced virus incidence by 35%, with a yield increase of 25% and a 24% improvement in fruit quality, when compared to the untreated control. Both mulching and the use of yellow sticky traps reduced the number of aphids trapped within the plots. In order to assist the development of capsicum cultivars resistant to PVY infections, a screening method was developed to determine susceptibility levels of a breeding population. Two rating procedures were investigated based on disease severity of the whole plant and on the fruit (chilli pods). The technique was effective in detecting small incremental increases in susceptibility within a breeding population, provided that an adequate positive selection pressure is applied. Using this technique breeders may be able to define a large breeding population to those parents exhibiting a genetic base most suitable for resistance development and eliminate those which exhibit low frequencies of resistance genes. Based on the results obtained, an integrated virus management strategy is suggested, including the elimination of virus sources and the use of cultural practices which facilitate a reduction in virus spread.
Cultivation of exotic and local mushroom species for commercial production.
(1995) Adey, Samantha.; Laing, Mark Delmege.
The project was undertaken primarily to determine the potential of growing Stropharia rugoso-annulata and Volvariella volvacea at a commercial level under South African conditions. Termitomyces umkowaani, an indigenous mushroom, was also investigated. Mushroom culturing, spawn production and optimizing fruiting substrates were determined. The feasibility of commercial and subsistence production in KwaZulu-Natal was investigated. Of the seven strains of S. rugoso-annulata purchased from CBS (Baarn, the Netherlands) that were tested, Strain 289.85 was the most vigorous. The best agar medium for culturing of S. rugoso-annulata was potato dextrose agar (PDA) followed by malt extract agar (MEA) and vegetable juice agar (V8). Maximum growth of the mushroom mycelium occurred from Day 7 to Day 14, as expected. The best spawn substrate tested was sorghum, and the best fruiting substrates tested were banana leaves and maize stover. As S. rugoso-annulata is a white-rot fungus capable of degrading lignin, this result was expected. S. rugoso-annulata grows best on substrates with a low C:N ratio. Strain 1665 of V. volvacea was the best of the three strains obtained from CBS. The best culture medium for V. volvariella was PDA amended with 1 % straw. V8 agar alone supported poor growth. In trials for suitable spawn and fruiting substrates for V. volvacea, banana leaves supported the best mycelial growth. Mycelium grown on V8 amended with 1% maize stover showed the greatest expansion on the substrates tested. A strain of T. umkowaani was isolated from a fresh basidiocarp collected in Pietermaritzburg, KwaZulu-Natal. Growth of T. umkowaani was most rapid on a Basal Medium but mycelial vigour was inferior to that of mycelium grown on Basal Medium amended with rabbit faeces, especially at the level of 2.5%. High levels of contamination occurred when more rabbit faeces was added to the Basal Medium. Microbial contamination was a major limiting factor to the productivity of this project. The primary sources of culture contamination were due to mites, inadequate sterile technique and a high level of Trichoderma spores in the Department due to an ongoing Trichoderma biocontrol project. Placing the bags of cultures on trays with the edges smeared with petroleum jelly effectively kept the mites away from the cultures. The contamination of a stock culture with Penicillium was solved with the use of Benomyl-amended agar media. Contamination problems in spawn production were the result of the inoculation process (especially when conducted by one person), the initial leaking of cotton wool caps, polypropylene bag seams and micropores and cracks in the walls of polypropylene bags. Treating the cotton wool in the caps with Busan 30A prevented the entry of contaminant bacteria and fungi through the caps. The problem of leaking seams of the bags was solved by using a longer heating period on the bag-sealer machine. SEM studies confirmed the presence of micropores and stress fractures in the walls of the bags used in this project. This problem can be solved by using high quality polypropylene with a reduced content of plasticiser. A systems analysis of exotic mushroom production, the process of mushroom production, steps in the process, sales and constraints in the South African context, and possible solutions are discussed. Spawn production by small growers is not economically feasible due to the capital required. Access to capital is a constraint of production in controlled environments but should not limit outdoor production. However, outdoor production is constrained by climatic requirements of the fungi. Based on optimum temperatures, GIS maps of KwaZulu-Natal were generated to display the potential areas for outdoor production. Overlap of suitable regions for production of S. rugoso-annulata and V. volvacea is limited. Outdoor production of V. volvacea will be limited to Northern coastal regions for only four months of the year. A computerized decision support system was developed to answer questions of feasibility of production to the mushroom growers, based on the requirements of the mushrooms. A current lack of marketing of speciality mushrooms is considered to be a major constraint to sales and therefore potential production, particularly for subsistence farmers.
The epidemiology and control of Leptosphaeria maculans cause of Crucifer Blackleg, in KwaZulu-Natal.
(1996) Laing, Mark Delmege.; Putter, Christoffel Antonie Johannes.; Rijkenberg, Fredericus Hermanus Johannes.
The perfect stage of Leptosphaeria maculans is reported for the first time in South Africa. Viable pseudothecia and pycnidia were found on dead, weathered tissue, sometimes in close association, whereas only pycnidia were found on live tissue. Some seedlots of imported cabbage seed were found to be internally infected with L. maculans at low levels and Alternaria brassicicola at higher levels. Fungicides iprodione (dicarboximide), triforine and propiconazole (sterol-biosynthesis inhibitor) eliminated both pathogens from infected seed. In a field trial of eight cabbage and two cauliflower cultivars, incidence of stem infection by L. maculans ranged from 16-80%. Two seedlots of the cabbage cultivar Gloria Osena differed in blackleg stem susceptibility. No correlation was found between stem lesion incidence and foliar infection counts of each cultivar, or stem lesion incidence and each cultivar's average days-to-harvest. In a second trial, incidence of stem infection ranged from 50% (Rotan) to 95% (Dynasty) in cabbage, and 64.2 to 96.6% in cauliflower cultivars. All Brussels sprouts and broccoli cultivars tested were highly susceptible. The cultivars of turnip and tyfon tested were observed to be immune to blackleg, whereas the swedes, Japanese radish, chou moullier and red cabbage cultivars tested were highly susceptible. No correlation was found between stem length and incidence of stem infection. Different seedlots within several cabbage and cauliflower cultivars differed in their blackleg susceptibility. A third cultivar trial with 10 replicates of four seedlots of one cabbage cultivar confirmed that different seedlots of a single cultivar may vary significantly in their susceptibility to blackleg. Benomyl was applied to cabbage at the seedling stage only, or at the seedling stage followed by field applications every 14 d. Relative to an untreated control, multiple applications of benomyl resulted in a 33% reduction in stem infection, a ten-fold reduction in plants killed and a 50% reduction in the proportion of non-harvestable heads, relative to an untreated control. Seedling treatment resulted in a lower infection level, a lower mortality rate and a greater mean head mass than those of the untreated control. However, none of these differences were statistically significant. In a debris degradation trial, more than 90% of buried debris (cabbage stems infected by L. maculans) had decomposed after 2.5 yr, whereas 80% of surface debris had decomposed over the same period. The susceptibilities of seedbed transplants (SBT) and container-grown seedlings (CGS) were compared using different forms of L. maculans inoculum. "Dunk" inoculation of SBT into a pycnidiosporial suspension resulted in a stem infection level of 50% greater than an uninocu1ated control. Contamination of seedbeds resulted in an infection level of 46%. "Dunk" inoculation of CGS resulted in infection level of 22%. When CGS were grown in contaminated trays an infection level of 33.4% resulted. Interplot interference ill the form of inoculum dispersal over a 1 m border was low (1.8 and 2.7% for SBT and CGS, respectively) . In a further trial examining the relationship of inoculum level and blackleg, a strong interaction was found between inoculation technique and inoculum level. Inoculation of field plots with infected debris was a more efficient technique than dipping seedlings into a pycnidiospore suspension prior to transplanting. Twenty nine blackleg epidemics were surveyed over 11 yr. Seedbed transplants (SBT) had been used in 83% of cases. Two cases (7%) had involved direct drilled seedlings (DDS). However, excess seedlings had been transplanted, making DDS epidemiologically equivalent to SBT. Three cases (10%) had involved container-grown seedlings (CGS) grown on mono cropped cabbage lands. Disease occurred in two patterns: in crops grown from SBT and DDS, blackleg occurred down the lines. In all CGS cases, disease occurrence was randomly patterned. In all cases, diseased debris was found in seedbeds and production fields. Disease spread in the field was limited to the two plants on either side of the initially infected plant, 1.3 m or less, suggesting that infection had resulted from splash dispersed pycnidiospores. The disease cycle was mono- or oligo cyclic but not polycyclic. Over a period of 6 yr, cabbage fields of 26 farms were each examined once for cruciferous weeds infected with L. maculans. No viable blackleg lesions were discovered on cruciferous weeds, suggesting that weeds play no role in the local crucifer blackleg pathosystem. A theory is proposed that windows of disease susceptibility open and shut during the different phenological stages of a crucifer's life, and that the susceptibility of different plant organs vary with the phenological state of the plant. It is also postulated that blackleg is a "low sugar disease". Disease incidence was lower in well fertilized cabbage plants than minimally fertilized plants. Organoleptic tests of cabbage cultivars correlated superior flavour and texture in cabbage with a high susceptibility to blackleg. An integrated management strategy is proposed, based on seed treatment with fungicides, the use of container-grown seedlings rather than seedbed transplants, a 3 yr rotation of crucifer lands with non-cruciferous crops, implementation of either deep-ploughing or accelerated biodegradation to eliminate debris, the development of higher levels of horizontal resistance to L. maculans in cruciferous vegetables, application of field fungicides in high risk areas (benzimidazoles or triazoles, or combinations), and the minimization of stress and optimization of host nutrition.
Epidemiology and management of grey leaf spot : a new disease of maize in South Africa.
(1996) Ward, John Michael Julian.; Cairns, Andrew Lawrence Patrick.; Rijkenberg, Fredericus Hermanus Johannes.; Laing, Mark Delmege.
Grey leaf spot is a relatively new fungal disease of maize in South Africa. It has become well established in the province of KwaZulu-Natal, and is capable of reducing grain yields by 20 to 60%. The disease is spreading to neighbouring provinces and countries. This study was conducted to establish solutions to the problem that could be easily implemented by maize farmers. Available literature was reviewed to establish the most appropriate epidemiologically based control measures that might be applicable in South Africa. Field trials were conducted to determine the effects of stubble and conventional tillage practices, cultivar susceptibility, fungicides, the correct time and frequency of fungicide treatment. and the financial benefits of fungicide treatment on grey leaf spot severity. The trials were evaluated for disease severity and grain yields. No commercial hybrids were identified to be resistant to grey leaf spot in the maize hybrid response to grey leaf spot trial. However, subsets of high-yielding hybrids less-susceptible to disease were identified - including PAN 6480, CRN 3584, SNK 2154 and PAN 6578. The most susceptible hybrids were identified to include RS 5206, PAN 6552, A 1849, PAN 6528 and PAN 6140. Fungicides containing carbendazim/flusilazole, were found to be most effective in controlling disease and increased maize yields. Hybrids such as RS 5206 and RS 5232 highly susceptible to disease and showed the highest grain yield response to fungicide treatment, whilst least-susceptible hybrids, such as PAN 6480, had the lowest response. The tillage trial aimed at management practices to reduce grey leaf spot indicated fungicides to be more effective in managing disease than tillage practices aimed at a reduction of initial inoculum. Trials on chemical control of grey leaf spot identified fungicides of the triazole and benzimidazole chemical groups to be effective in controlling disease, but only combination products of these chemical groups, were registered, in support of the pathogen resistance strategy. Products registered were carbendazim/flusilazole, carbendazim/flutriafol and carbendazim/difenoconazole. The frequency and timing of fungicide applications for the control of grey leaf spot in maize studies identified spray treatments initiated when disease had progressed to the basal five leaves and, before the exponential phase of the epidemic, provided the most effective disease control and concomitant high grain yields. Further spray treatments were necessary with early disease infections, in order to provide disease control until crop physiological maturity. The final study on the economic benefits of fungicide treatment of grey leaf spot in maize in KwaZulu-Natal indicated that the highest added yield response was not necessarily the best parameter to justify fungicide treatment. Rather, the expected added profit was a better parameter. In this study the highest added profits were R1 400 ha(-1) from the triple-spray programme in 1993/94 and R439 ha(-1) from a single-spray in 1992/93. The optimum treatment choice depended on the individual's risk-return preferences, which reflect his level of risk-aversion. An integrated approach using tillage practices, crop rotations, hybrids less- susceptible to the pathogen and the judicious use of fungicides is likely to be the most successful in controlling the disease. In the long term, the cornerstone of the integrated approach will be the development and use of hybrids resistant to the disease.
Ultrastructure, cytochemistry and immunocytochemistry of the interaction between wheat (Triticum aestivum) and leaf rust (Puccinia Recondita f.sp. tritici)
(1996) Hu, Guanggan.; Rijkenberg, Fredericus Hermanus Johannes.
The development of infection structures, derived from urediospores of Puccinia recondita f.sp. tritici in near-isogenic lines of susceptible and resistant wheat, and in non-hosts, viz. maize, oat, sorghum and barley, was examined by fluorescence microscopy and scanning electron microscopy (SEM). The infection structure formation on and in five cereal species follows a similar pattern. In sorghum, fungal development is arrested at the stage of substomatal vesicle formation, while, in maize, most fungal structures collapse during the stage of primary hypha development. On the other hand, in wheat, barley and oat, the fungus forms many branched infection hyphae and haustorial mother cells. There were no significant structural and numerical differences in infection structure development between susceptible and resistant wheat lines. The ultrastructure of intercellular hyphae and D-haustoria of P. recondita f.sp. tritici, and the host response to haustorial invasion, was investigated. The intercellular hyphae share common characteristics with other uredial stage rust fungi. Anastomosis was observed between intercellular hyphae. Two nucleoli were frequently observed in a single nucleus in the haustorium, indicating possible nuclear fusion between the two nuclei in D-haustoria of this fungus. The close association of host organelles, such as the nucleus, Golgi bodies, endoplasmic reticulum , vesicles and mitochondria, with the developing haustorium, was described. The investigation of urediospore formation of P. recondita f.sp. tritici on wheat leaves by SEM and transmission electron microscopy (TEM) showed that one or more protuberances arise sympodially from several different loci on the distal surface of a basal cell, each protuberance developing into a urediospore. At the same site at which one urediospore formed previously, at least one other urediospore initial can form subsequently. A study of the cytochemistry of the interaction between wheat and P. recondita f.sp. tritici, using various enzyme- and lectin-conjugated gold probes, was conducted. This research provided additional information on the nature and composition of the walls of fungal hyphae, the haustorial mother cell, the haustorial neck, the haustorial body and the extrahaustorial matrix. Cellulose, the major component of the host cell wall, was not detect.ed in the extrahaustorial matrix and in the host tubules associated with the invaded haustorium. The composition of walls of the haustorial body of P. recondita f .sp. tritici appears to change as the haustorium matures. The study identified the existence of mannose/glucose, galactose, N-acetylgalactosamine and fucose residues in the extrahaustorial matrix. An antiserum raised against the purified 33 kDa wheat β-1 ,3-glucanase was used to investigate the subcellular localization of the enzyme in P. recondita f.sp. tritici-infected wheat leaves by means of a post-embedding immunogold labelling technique. In the compatible interaction, β-1,3-glucanase was demonstrated to accumulate predominantly in the haustorial wall and extrahaustorial matrix. In the incompatible interaction, strong labelling for β- 1 ,3-glucanase was found in host cell wall appositions, extracellular matrix in the intercellular space, and in electron-dense structures of host origin which only occurred in the incompatible interaction. Using anti-zeatin riboside and anti-isopentenyladenosine antibodies in post-embedding immunocytochemical procedures, cytokinins were localized at the ultrastructural level in P. recondita f .sp. tritici-infected wheat leaves. The sites where cytokinins accumulate were not significantly different between the compatible and incompatible interactions. The cytokinins are mainly present in the fungal cytoplasm of the intercellular hyphal cell, the haustorial mother cell, the haustorial body and extrahaustorial matrix, indicating that cytokinins, primarily of fungal origin, are associated with the nutrient translocation in this host-fungus interaction.
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.
Studies on ear rot and grey leaf spot of maize in South Africa.
(1997) Nowell, David C.; Rijkenberg, Fredericus Hermanus Johannes.; Laing, Mark Delmege.
In recent years there have been economically important epidemics of both Stenocarpella ear rot and grey leaf spot (GLS) in South Africa. These epidemics have adversely affected the grain yield and quality of the maize harvested. Maize researchers and breeders have had to re-assess the importance of maize disease in South Africa and make the necessary adjustments to their programmes. Literature reviews were undertaken on both Stenocarpella ear rot and GLS to provide the necessary background of technical information to conduct research under local conditions into these disease problems, and to assist in interpretation of results of experiments. A novel method of inoculating milled Stenocarpella infected ears into the whorls of maize plants (about 2 weeks before 50% anthesis) was developed to provide consistent inoculum pressure and increased ear rot. This inoculation method was practical, efficient, reliable, consistent and cheap to implement. Commercial organisations could use this inoculation method to inoculate a large number of plants per day, allowing for improved screening of breeding material and hybrids. Ear rot assessment methods, and researchers' ability to assess ear rot, were tested under South African conditions. The accuracy of the different methods tested varied considerably, particularly when there was a high level of ear rot that could not be seen without shelling the grain. Each method could be used in a maize breeding programme, depending upon the desired levels of accuracy and time taken using the given method. Researcher's ability to assess ear rot varied considerably and accuracy was correlated with the number of years experience in maize research. Grain colour affected the researcher's ability to accurately assess ear rot severity. Yellow-grained maize was more difficult to assess for ear rot than white-grained maize. Hybrid response to Stenocarpella ear rot infection was difficult to interpret owing to a significant interaction with the environment. Hybrid ear rot response was non-linear in nature. Normal methods of presenting disease data and classifying hybrids in resistance response categories were not successful. Non-linear regression analysis has to be used to do this. However, it is important that ear rot data be presented in a way that farmers can utilise the information. Pre-flower stress predisposes maize hybrids to ear rot infection. Hybrids that normally exhibited good levels of resistance to Stenocarpella ear rot may become severely colonised if drought stress occurs in the four weeks prior to flowering. This environmental interaction makes ear rot resistance breeding and the interpretation of results difficult. The incidence of maize ear rot was widely considered to increase with increased plant density. Experiments over three seasons in South Africa have shown that is not true under certain environmental conditions. In specific hybrids, plant densities of less than 50 000 plants ha"1 exhibited a higher incidence and severity of ear rot than plant densities greater than 50 000 plants ha(-1). The hybrids that usually responded in this manner were more susceptible to ear rot than the other hybrids. Generally, ear rot increased with increased plant densities over 50 000 plants ha(-1). The mechanisms and reasons for this could not be determined. Fungicide trials and regression analysis of hybrid yield trials over a two years period, at two locations in KwaZulu-Natal, showed that grain yield losses due to GLS infection were at least 13%. Severity of GLS was consistently higher at Cedara than at Greytown. Economic losses at Cedara ranged from Rl 919 - R2 278 ha(-1) and at Greytown from Rl 554 - Rl 726 ha(-1). Predicted hybrid losses ranged from R836 - R2 621 ha(-1) (13% - 37%), depending upon the level of inherent GLS resistance. Hybrid response to Cercospora zeae-maydis infection was linear in nature and hybrids could be categorised into response categories. Large differences in GLS resistance could be found between commercial hybrids. However, the current levels of GLS resistance in hybrids does not eliminate yield loss under high GLS inoculum levels, and fungicide application was economically justified on most hybrids. Newly released hybrids show increased levels of GLS resistance. The application of systemic fungicides to GLS-susceptible maize was highly effective in controlling GLS and increasing yield substantially. The most effective fungicides belonged to the triazole and benzimidazole group of fungicides. Protectant fungicides were not as effective as systemic fungicides. Copper-based fungicides were phytotoxic to maize in two seasons and at both locations. Fungicide mixtures of the two groups active against GLS are being used on commercially. The effectiveness of fungicides did not vary over location or hybrids, but was influenced by inoculum pressure. Effective control strategies have been implemented to control both Stenocarpella ear rot and GLS in South Africa. Crop rotation, the selection of the more ear rot and GLS-resistant hybrids, and the judicious use of fungicides has reduced the levels of both diseases to manageable levels. An integrated control strategy is needed to control these diseases and efforts are being made to educate farmers to this effect. Maize pathological research now enjoys a greater emphasis than it did in the early-1980s.
Control of insect-transmitted viruses in cucurbit crops in KwaZulu- Natal.
(1998) Cradock, Kenwyn Roan.; Da Graca, John Vincent.; Laing, Mark Delmege.
The production of cucurbits (Cucurbitaceae) in KwaZulu-Natal faces the constant threat of viral diseases. These can be so severe as to severely limit or prevent production in the latter part of the growing season (December-April). The important viruses in this regard are zucchini yellow mosaic potyvirus (ZYMV), watermelon mosaic 2 potyvirus (WMV2), watermelon mosaic potyvirus - Morocco strain (WMV-M), papaya ringspot potyvirus - type W (PRSV-W), cucumber mosaic cucumovirus (CMV), and squash mosaic comovirus (SqMV). The potyviruses and CMV are vectored by aphids (Homoptera: Aphididae) and SqMV is vectored by cucumber beetles (Coleoptera: Coccinelidae). PRSV and SqMV were found to be absent from the region, while CMV was found not to be a serious threat to cucurbit production. ZYMV, WMV2 (now confirmed to occur in South Africa) and WMV-M are the major viral pathogens of cucurbits in KwaZulu-Natal. The distribution of these viruses and methods for their control were investigated. Investigations of aphid morphology using the scanning electron microscope were undertaken to determine if taxonomic studies could be conducted using this form of microscopy. The best form of specimen preparation was the cryo-fixation technique, which resulted in less collapse of the body wall and general damage to the specimen when compared to the critical point drying technique. Due to the lack of mobility of the specimen while viewing, this form of microscopy is rejected as a means of identifying aphids to the species level. ZYMV was found to occur in a number of weed species (Galinsoga parviflora, Malva parviflora, Amaranthus sp., Solarium spp.), which could serve as reservoirs of virus. WMV-M and CMV were also found in some weed species. All tests for the potyviruses and SqMV were done using the double-antibody sandwich (DAS) ELISA technique. CMV was tested for using indirect ELISA tests. A third of the plants tested were found to be infected with more than one virus which could have implications for disease severity. Disease severity was found to increase at about midway through the growing season (December- January). This was concurrent with a massive increase in the general aphid population in the experimental area. As no aphids were seen on the cucurbits in the fields, these vectors are believed to be transient inhabitants of the crop at first testing and then rejecting the plants as a food source. All control measures applied in the trial were aimed at reducing the numbers of aphids in the plots. Aphids were trapped using yellow sticky traps. Cucurbita pepo (zucchini) was used in the trials due to its bush growth habit and good virus symptom expression. The success of the treatments was determined by monitoring the numbers of aphids present in the plots, and the use of a rating scale which assessed the severity of virus disease in the plots. The two best treatments were the white reflective mulch and the straw mulch. In the cultivar trial which assessed ten different cultivars for their virus resistance/tolerance. The best three cultivars were 'SQ 229', 'Puma', and 'SQ 228'. 'SQ 229' and 'SQ 228' were withdrawn from the market by the seed company for unknown reasons. From the results obtained from these investigations, a disease management programme can be suggested. All cucurbit crops should be grown over a white reflective mulch, drip irrigation should be used to reduce agitation of the plants which could unnecessarily disturb feeding aphids, and a resistant or tolerant cultivar should be used in the latter half of the growing season. The effectiveness of any treatment can be assessed by comparing the number of aphids caught with the number caught in the control plots.

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