Masters Degrees (Plant Pathology)

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Biological control of the grapevine trunk disease pathogens : pruning wound protection
(Stellenbosch : Stellenbosch University, 2008-12) Kotze, Charl; Fourie, P. H.; Van Niekerk, J. M.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
In recent years, several studies have conclusively shown that numerous pathogens, including several species in the Botryosphaeriaceae, Phomopsis, Phaeoacremonium, as well as Phaeomoniella chlamydospora and Eutypa lata, contribute to premature decline and dieback of grapevines. These pathogens have the ability to infect grapevines through pruning wounds, which leads to a wide range of symptoms developing that includes stunted growth, cankers and several types of wood necrosis. Pruning wounds stay susceptible for 2 to 16 weeks after pruning and sustained levels of pruning wound protection is therefore required. The aims of this study were to (i) evaluate the ability of several biological agents to protect pruning wounds, (ii) characterise unknown Trichoderma strains and identify their modes of action and (iii) determine the optimal time of season for biological agent application. Several biological agents were initially evaluated in a laboratory for their antagonism against trunk disease pathogens. The best performing control agents were tested in a field trial conducted on Merlot and Chenin blanc vines in the Stellenbosch region. Spurs were pruned to three buds and the fresh pruning wounds were treated with benomyl as a control treatment, Trichoderma-based commercial products, Vinevax® and Eco77®, Bacillus subtilis, and Trichoderma isolates, USPP-T1 and -T2. Seven days after treatment the pruning wounds were spray inoculated with spore suspensions of four Botryosphaeriaceae spp. (Neofusicoccum australe, N. parvum, Diplodia seriata and Lasiodiplodia theobromae), Eutypa lata, Phaeomoniella chlamydospora and Phomopsis viticola. After a period of 8 months the treatments were evaluated by isolations onto potato dextrose agar. Trichodermabased products and isolates in most cases showed equal or better efficacy than benomyl, especially USPP-T1 and -T2. Moreover, these isolates demonstrated a very good ability to colonise the wound tissue. The two uncharacterised Trichoderma isolates (USPP-T1 and USPP-T2), which were shown to be highly antagonistic toward the grapevine trunk disease pathogens, were identified by means of DNA comparison, and their ability to inhibit the mycelium growth of the trunk disease pathogens by means of volatile and non-volatile metabolite production studied. The two gene areas that were used include the internal transcribed spacers (ITS 1 and 2) and the 5.8S ribosomal RNA gene and the translation elongation factor 1 (EF). The ITS and EF sequences were aligned to published Trichoderma sequences and the percentage similarity determined and the two Trichoderma isolates were identified as Trichoderma atroviride. The volatile production of T. atroviride isolates was determined by placing an inverted Petri dish with Trichoderma on top of a dish with a pathogen isolate and then sealed with parafilm. Trichoderma isolates were grown for 2 days on PDA where after they were inverted over PDA plates containing mycelial plugs. The inhibition ranged from 23.6% for L. theobromae to 72.4% for P. viticola. Inhibition by non-volatile products was less than for the volatile inhibition. Inhibition ranged from 7.5% for N. parvum to 20.6% for L. theobromae. In the non-volatile inhibition USPP-T1 caused significantly more mycelial inhibition than USPP-T2. The timing of pruning wound treatment and subsequent penetration and colonisation of the wound site was also determined. One-year-old canes of the Shiraz and Chenin blanc cultivars were grown in a hydroponic system, pruned and spray treated with a spore suspension of Trichoderma atroviride (USPP-T1) as well as a fluorescent pigment. On intervals 1, 3, 5 and 7 days after treatment, the distal nodes were removed and dissected longitudinally. From the one half, isolations were made at various distances from the pruning surface, while the other half was observed under ultra-violet light to determine the depth of fluorescent pigment penetration. Shortly after spray-inoculation of a fresh pruning wound, Trichoderma was isolated only from the wound surface and shallow depths into the wound (2 to 5 mm). One week after inoculation, Trichoderma was isolated at 10 mm depths, and after 2 weeks, at 15 mm depths. Fluorescent pigment particles were observed to a mean depth of 6 mm, which suggests that initial isolation of Trichoderma at these depths was resultant of the physical deposition of conidia deeper into the pruning wound tissue, whereas the isolation of Trichoderma from deeper depths might be attributed to colonisation of grapevine tissue. In a vineyard trial, fluorescent pigment was spray-applied to pruning wounds of Shiraz and Chenin blanc grapevines during July and September at intervals 0, 1, 3, 7 and 14 days after pruning. One week after treatment, the distal nodes were removed and dissected longitudinally. Each half was observed under UV light and the pigment penetration measured. For Chenin blanc and Shiraz, July pruning wounds showed significant deeper penetration of the pigment than pruning wounds treated in September. Moreover, pruning wounds made in September showed pigment particles in longitudinal sections up to 1 day after pruning, whereas wounds made in July showed pigment particles up to 3 days in the xylem vessels. These findings suggest that the best time for application of a biological control agent should be within the first 24 hours after pruning.
Characterisation and management of trunk disease-causing pathogens on table grapevines
(Stellenbosch : Stellenbosch University, 2006-04) Bester, Wilma; Fourie, P. H.; Crous, P. W.; Stellenbosch University. Faculty of Agrisciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Phaeomoniella chlamydospora, Eutypa lata, Phomopsis, Phaeoacremonium, and Botryosphaeria spp. are important trunk disease pathogens that cause premature decline and dieback of grapevine. Previous research has focused primarily on wine grapes and the incidence and symptomatology of these pathogens on table grapes were largely unknown. A survey was therefore conducted to determine the status and distribution of these pathogens and associated symptoms in climatically diverse table grape growing regions. Fifteen farms were identified in the winter rainfall (De Doorns, Paarl and Trawal) and summer rainfall (Upington and Groblersdal) areas. Samples were taken in July and August 2004 from Dan-ben-Hannah vineyards that were 8 years and older. Distal ends of arms were removed from 20 randomly selected plants in each vineyard. These sections were dissected and isolations were made from each of the various symptom types observed: brown or black vascular streaking, brown internal necrosis, wedge-shaped necrosis, watery necrosis, esca-like brown and yellow soft wood rot, as well as asymptomatic wood. Fungal isolates were identified using molecular and morphological techniques. Pa. chlamydospora was most frequently isolated (46.0%), followed by Phaeoacremonium aleophilum (10.0%), Phomopsis viticola (3.0%), Botryosphaeria obtusa (3.0%), B. rhodina (2.2%), B. parva (2.0%), Fusicoccum vitifusiforme (0.6%), B. australis, B. dothidea and an undescribed Diplodia sp. (0.2% each), while E. lata was not found. Most of these pathogens were isolated from a variety of symptom types, indicating that disease diagnosis can not be based on symptomatology alone. Pa. chlamydospora was isolated from all areas sampled, although most frequently from the winter rainfall region. Pm. aleophilum was found predominantly in Paarl, while P. viticola only occurred in this area. Although B. obtusa was not isolated from samples taken in De Doorns and Groblersdal, it was the most commonly isolated Botryosphaeria sp., being isolated from Upington, Paarl and Trawal. B. rhodina occurred only in Groblersdal and B. parva in Paarl, Trawal and Groblersdal, while B. australis was isolated from Paarl only. The rest of the isolates (33%) consisted of sterile cultures, Exochalara, Cephalosporium, Wangiella, Scytalidium, Penicillium spp. and two unidentified basidiomycetes, which were isolated from five samples with yellow esca-like symptoms from the Paarl area. These findings clearly illustrate that grapevine trunk diseases are caused by a complex of fungal pathogens, which has serious implications for disease diagnosis and management. Protection of wounds against infection by any of these trunk disease pathogens is the most efficient and cost-effective means to prevent grapevine trunk diseases. However, previous research on the effectiveness of chemical pruning wound protectants has mostly focused on the control of Eutypa dieback only. Fungicide sensitivity studies have been conducted for Pa. chlamydospora, P. viticola and Eutypa lata, but no such studies have been conducted for the pathogenic Botryosphaeria species from grapevine in South Africa. Ten fungicides were therefore tested in vitro for their efficacy on mycelial inhibition of the four most common and/or pathogenic Botryosphaeria species in South Africa, B. australis, B. obtusa, B. parva and B. rhodina. Iprodione, pyrimethanil, copper ammonium acetate, kresoxim-methyl and boscalid were ineffective in inhibiting the mycelial growth at the highest concentration tested (5 μg/ml; 20 μg/ml for copper ammonium acetate). Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole were the most effective fungicides with EC50 values for the different species ranging from 0.36-0.55, 0.07-0.17, 0.07-1.15 and 0.04-0.36 μg/ml, respectively. These fungicides, except prochloraz manganese chloride, are registered on grapes in South Africa and were also reported to be effective against Pa. chlamydospora, P. viticola and E. lata. Results from bioassays on 1-year-old Chenin Blanc grapevine shoots indicated that benomyl, tebuconazole and prochloraz manganese chloride were most effective in limiting lesion length in pruning wounds that were inoculated with the Botryosphaeria spp after fungicide treatment. The bioassay findings were, however, inconclusive due to low and varied re-isolation data of the inoculated lesions. Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole can nonetheless be identified as fungicides to be evaluated as pruning wound protectants in additional bioassays and vineyard trials against Botryosphaeria spp. as well as the other grapevine trunk disease pathogens.
Characterisation of pathogens associated with trunk diseases of grapevines
(Stellenbosch : Stellenbosch University, 2004-04) Van Niekerk, Jan Marthinus; Crous, P. W.; Fourie, P. H.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology .
ENGLISH ABSTRACT: In an attempt to combat some of the pathogens that are associated with trunk diseases and disorders of grapevines, research in this thesis focused on the taxonomy and pathological aspects of Coniellai/Pilidiella, Botryosphaeria and Phomopsis spp. Previously, conidial pigmentation was used to separate Pilidiella from Coniella. Recently, however, the two genera have been regarded as synonymous, with the older name, Coniella, having priority. The most important species in the Coniellai/Pilidiella complex of grapevines is C. diplodiella (Speg.) Petr. & Syd., the causal organism of white rot of grapevines. Previous studies found it difficult to distinguish between C. diplodiella and C. fragariae (Oudem.) B. Sutton, which is known to occur in soil and caused leaf diseases of Fragaria and Eucalyptus. Both these species have previously been reported from South Africa. None of the reports on C. diplodiella could be scientifically substantiated; therefore it is still a quarantine organism. However, this status has been questioned. Based on sequence analyses of the internal transcribed spacer region (ITS 1, ITS 2), 5.8S gene, large subunit (LSU) and elongation factor 1- α gene (EF l- α) from the type species of Pilidiella and Coniella, Coniella was separated from Pilidiella, with the majority of taxa residing in Pilidiella. Pilidiella is characterised by species with hyaline to pale brown conidia (avg. length: width >1.5), with Coniella having dark brown conidia (avg. length: width ≤1.5). Pilidiella diplodiella, previously C. diplodiella, causal organism of white rot of grapevines, was shown to be an older name for C. petrakii. This fungus is present in South Africa and is therefore no longer of quarantine importance. Based on analyses of the histone (H3) gene sequences of isolates in the P. diplodiella species complex, P. diplodiella was separated from a newly described species, P. diplodiopsis. A new species, P. eucalyptorum, is proposed for isolates formerly treated as C. fragariae, associated with leaf spots of Eucalyptus spp. This species clustered basal to Pilidiella, and may represent yet a third genus within this complex. Pilidiella destruens was newly described as anamorph of Schizoparme destruens, which is associated with twig dieback of Eucalyptus spp. in Hawaii. The genus Botryosphaeria Ces. & De Not. are known to be cosmopolitan, with broad host ranges and geographical distributions. Several saprotrophic species have been reported from grapevines, while others are severe pathogens of this host. These species include B. dothidea (Moug.: Fr.) Ces. & De Not., B. parva Pennycook & Samuels, B. obtusa (Schwein.) Shoemaker, B. stevensii Shoemaker, B. lutea A.J.L. Phillips and B. ribis Grossenb. & Duggar. Species reported from South Africa as grapevine pathogens are B. obtusa, B. dothidea, B. ribis and B. vitis (Schulzer) Sacco. In the present study, morphological, DNA sequence data (ITS 1, 5.8S, ITS 2 and EFI-α) and pathological data were used to distinguish 11 Botryosphaeria spp. associated with grapevines from South Africa and other parts of the world. Botryosphaeria australis, B. lutea, B. obtusa, B. parva, B. rhodina and a Diplodia sp. were confirmed from grapevines in South Africa, while Diplodia porosum, Fusicoccum viticlavatum and F. vitifusiforme were described as new species. Although isolates of B. dothidea and B. stevensii were confirmed from grapevines in Portugal, neither of these species, nor B. ribis, were isolated in this study. All grapevine isolates from Portugal, formerly presumed to be B. rib is, are identified as B. parva based on EF1-α sequence data. Artificial inoculations on grapevine shoots showed that B. australis, B. parva, B. ribis and B. stevensii are more virulent than the other species studied. The Diplodia sp. collected from grapevine canes was identified as morphologically similar, but phylogenetically distinct from D. sarmentorum, while D. sarmentorum was confirmed as anamorph of Otthia spiraeae, the type species of the genus Otthia (Botryosphaeriaceae). A culture identified as O. spiraeae clustered within Botryosphaeria, and is thus regarded as a probable synonym. These findings confirm earlier suggestions that the generic concept of Botryosphaeria should be expanded to include genera with septate ascospores and Diplodia anamorphs. The genus Phomopsis (Sacc.) Bubak contains many species that are plant pathogenic or saprotrophic. Ten species are known from grapevines. However, only two have been confirmed as being pathogenic, namely P. viticola (Sacc.) Sacc., causal organism of Phomopsis cane and leaf spot and P. vitimegaspora Kuo & Leu (teleomorph Diaporthe kyushuensis Kajitani & Kanem.), causal organism of swelling arm disease of grapevines. P. amygdali (Delacr.) 1.1. Tuset & M.T. Portilla, a known pathogen from Prunus sp., was shown to be a possible pathogen of grapevines in a previous study. D. perjuncta Niessl. causes bleaching of dormant canes only and is therefore of little importance as a grapevine pathogen. Recently a number of Phomopsis isolates were obtained from grapevines in the Western Cape province of South Africa. Isolations were made from Phomopsis-like symptoms, pruning wounds and asymptomatic nursery plants. These isolates showed great variation in morphology and cultural characteristics. Earlier taxonomic treatments of Phomopsis, based species identification on host specificity, cultural characteristics and morphology. Recent studies have indicated that these characteristics can no longer be used to distinguish species of Phomopsis due to wide host ranges and morphological plasticity of some species. The use of anamorph/teleomorph relationships in species identification is also untenable, since Diaporthe teleomorphs have only been described for approximately 20% of the known Phomopsis species. In this study morphological data, DNA sequences (ITS-I, 5.8S, ITS-2) and pathogenicity data were combined to distinguish Phomopsis spp. from grapevines. Fifteen species of Phomopsis were delineated by phylogenetic analysis of ITS sequence data. Diaporthe helianthi, a sunflower pathogen, was reported from grapevines for the first time, with a further six, unknown species also distinguished. Three different clades contained isolates previously identified as D. perjuncta. Based on type studies, it appeared that the name D. viticola was available for collections from Portugal and Germany, a new species, D. australafricana, was proposed for South African and Australian isolates, formerly treated as D. perjuncta or D. viticola. An epitype specimen and culture were designated for D. perjuncta. This species was distinguished from D. viticola and D. australafricana based on morphology and DNA phylogeny. Artificial inoculations of green grapevine shoots indicated that, of the species tested, P. amygdali, a known pathogen of peaches in the USA, and P. viticola were the most virulent.
Genetic diversity in Plasmopara viticola in South Africa
(Stellenbosch : Stellenbosch University, 2007-03) Koopman, Trevor A; McLeod, Adele; Fourie, P. H.; Stellenbosch University. Faculty of Agriscience. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Downy mildew, caused by the obligate pathogen Plasmopara viticola, is a very destructive grapevine disease. The asexual phase (sporangia) of the pathogen has long been viewed as the life cycle stage that is most important in causing expansion of epidemics. Contrarily, the role of the sexual phase (oospores) has primarily been viewed as only providing the initial primary inoculum of the epidemic at the start of the season. However, population genetic studies in Europe have challenged these long standing epidemiological views. Downy mildew is mainly controlled through the application of fungicides, since no commercially acceptable resistant cultivars are available. The use of reliable high throughput in vitro resistance screening methods is very important for identifying new sources of resistance, as well as for mapping of quantitative trait loci (QTLs) involved in downy mildew resistance. Resistance screenings also require the use of effective longterm pathogen storage methods, since it allows the continual use of the same well characterised P. viticola isolates in different resistance screenings over seasons. The first main aim of this study was to investigate the population genetic structure of P. viticola populations in South Africa in two vineyards. The second aim was to determine whether an in vitro leaf disk method is a reliable and reproducible resistance screening method for determining downy mildew resistance of grapevine seedlings. The third aim was to evaluate long-term storage techniques for P. viticola isolates. The population genetic structure of P. viticola was investigated m two consecutive grape growmg seasons in an organically managed and a conventional fungicide sprayed vineyard. The study showed that population differentiation between the two vineyards was low (0.004 and 0.016) in both growing seasons, suggesting one metapopulation. New genotypes (12% to 74%) contributed to the epidemic throughout the growing seasons in both years and vineyards. The epidemic in both years and vineyards were dominated by one or two genotypes, which contributed between 14% and 67% through asexual reproduction to the epidemic. The remaining genotypes showed low levels of asexual reproduction, with most genotypes never being able to reproduce asexually. Ten genotypes were able to survive asexually from one season to the next. Moreover, the predominant genotype in the organically grown vineyard during 2004/05 survived asexually to the next season, where it also dominated the epidemic. Evaluation of an in vitro leaf disk method showed that the method was a reliable and reproducible method for screening the downy mildew resistance of the progeny of a Regent x Red Globe cross. Spearman correlation analyses revealed a moderate to high (0.64 to 0.82) correlation between three screening trails that were conducted over two growing seasons. However, the percentage seedlings that belonged to the different OIV 452 rating classes differed between the third (2005/06) and the first two (2004/05) resistance screening trials. This difference was statistically supported by one-way analysis of variance of rank means of these screenings, as well as Chi-square test of the screening x rating scale contingency table. This discrepancy indicates the importance of the inclusion of tolerant and sensitive reference seedlings, as well as the parents of the cross in each screening trial. Evaluation of different long-term storage methods for P. viticola showed that the pathogen was best stored as lesions. Successful storage methods included the storage of whole leaves with sporulating lesions in sealed Petri dishes, or the storage of small leaf lesion fragments within 2 ml centrifuge tubes at -20 and -80°C. Viability testing of these storage methods after a period of 6 (leaves within Petri plates) and 1 7 months (lesions within centrifuge tubes) showed that the pathogen remained viable for these periods, although the viability of sporangia were reduced.
Mancozeb rainfastness and residue thresholds for control of Venturia inaequalis
(Stellenbosch : Stellenbosch University, 2016-03) Rossouw, Cornelis Johannes; McLeod, Adele; Fourie, P. H.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH SUMMARY: An important apple disease world-wide is Venturia inaequalis that causes scab-like lesions on fruit and defoliation of trees. In South Africa, the disease is mainly managed through fungicide sprays that mostly consist of the contact fungicide mancozeb. The volume and intensity of rainfall can influence the efficacy and persistence of mancozeb depositions. Mancozeb spray deposition can be assessed through expensive quantification of Manganese-ion residues, or a cost effective fluorescent pigment of which the coverage is accessed using photomacrography image analyses. The biological efficacy of quantitative mancozeb depositions can be determined through the development of benchmark models. For V. inaequalis, this is challenging since in vitro production of inoculum is difficult, and leaf lesions are slow to develop and not amendable to quantification through image analyses. The study showed that a yellow fluorescent pigment was a suitable tracer for five different mancozeb formulations (Dithane M-45 800 WP NT, Mancozeb 800 WP, Pennfluid, Ventum 800 WP, Vondozeb) since a good Pearson’s correlation (r = 0.779) existed between fluorescent particle coverage (FPC%) and mancozeb residues (Mn-ion (mg/kgDW)).The particle size ranges of Dithane M-45 800 WP NT and Ventum 800 WP were significantly smaller than those off the other formulations, but this did not result in differences in Mn-ion residues realized on apple leaves for WP formulations. The Pennfluid SC formulation deposited markedly less mancozeb than the WP formulations, due to a lower active ingredient label rate used. Rainfastness was evaluated for the Dithane M-45 800 WP NT and Ventum 800 WP formulations, and Ventum 800 WP combined with a sticker-spreader adjuvant Nu-Film P. Simulated rain applied to apple seedlings at a constant rainfall intensity of 5 mm/h at five different rainfall volumes (0, 1, 5, 10 and 15 mm) resulted in no significant differences between the three treatments, based on FPC% and Mn-ion concentrations. Although a good correlation (r = 0.726 to 0.783) existed between FPC% and Mn-ions, the response of FPC% and Mn-ion differed somewhat as was evident from the slopes of exponential regression models, showing slower initial loss in FPC% than for Mn-ions and a markedly larger predicted loss by the model’s asymptotic value (61.4% and 32.2%, respectively). A significant loss (11.95%) in mancozeb residue occurred after applying 1mm of rain, but no significant differences in losses (26.32 to 31.67%) occurred after applying 5 to 15mm of rain. Benchmark development for mancozeb deposition for the control of V. inaequalis involved apple seedling leaves being treated with a concentration range of mancozeb and fluorescent pigment (0, 0.15 ×, 0.3 ×, 0.45 ×, 0.6 × and 1.0 ×), followed by inoculation with conidia harvested from naturally infected orchard leaves. Venturia inaequalis control was assessed using a basic fuschin based staining technique and visual assessment. The staining technique was useful for quantifying infection within 6 days, but underestimated percentage control relative to the visual assessment of lesions after 3-4 weeks. Complete control was observed for all mancozeb concentrations based on visual lesion assessment. No function could thus be fitted to deposition quantity data (0.29 to 8.28 FPC% values) versus disease control (staining or visual assessment). The cellophane agar plate technique was optimized for the in vitro production of V. inaequalis conidium inoculum that can be used in future infection studies. After 1 week, optimum spore production (1.59× 106 conidia/ml) and viability (± 85%) were observed that were significantly higher than at weeks 2 to 4. The study provided valuable information for the assessment of mancozeb deposition, mancozeb rainfastness, and for benchmark model development with regards to the rapid quantification of lesions on leaves and the in vitro production of V. inaequalis inoculum. The yellow fluorescent pigment can now be used as an excellent cost effective tracer for mancozeb deposition of various formulations on apple seedling leaves, and will also be helpful in identifying trends on the effect of rain on the persistence of mancozeb formulations. This has increased research capacity towards evaluating the rainfastness of fungicides using simulated rain, and orchard trials for accessing the effect of spray volumes and machines through fluorescent pigment deposition. Although a benchmark model could not be developed, the development of a model in future will be more feasible since a rapid staining technique for quantification of V. inaequalis disease severity was identified that will just have to be optimized further. Secondly, a cellophane agar plate technique, and isolates with high spore production capacity for axenic conidial production will further facilitate model development.
Molecular detection of Phaeomoniella chlamydospora in grapevine nurseries
(Stellenbosch : Stellenbosch University, 2005-04) Retief, Estianne; Fourie, P. H.; McLeod, Adele; Stellenbosch University. Faculty of Agrisciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Phaeomoniella chlamydospora is the main causal organism of Petri disease, which causes severe decline and dieback of young grapevines (1-7 years old) and also predisposes the wood for infection by other pathogens. Knowledge about the epidemiology and especially inoculum sources of this disease is imperative for subsequent development of management strategies. Through isolation studies it was shown that Pa. chlamydospora is mainly distributed through infected propagation material in South Africa. However, the infection pathways and inoculum sources in grapevine nurseries are still unclear. The only existing method to detect this pathogen in various media is by means of isolation onto artificial growth media. This has proven to be problematic since this fungus is extremely slow growing (up to 4 weeks from isolation to identification) and its cultures are often over-grown by co-isolated fungi and bacteria before it can be identified. The aim of this study was (i) to develop a protocol for the molecular detection of Pa. chlamydospora in grapevine wood, and (ii) to use this protocol along with others, to test different samples (water, soil, rootstock and scion cuttings and callusing medium) collected from nurseries in South Africa at different nursery stages for the presence of Pa. chlamydospora. A protocol was developed and validated for the molecular detection of Pa. chlamydospora in grapevine wood. Firstly, several previously published protocols were used to develop a cost-effective and time-efficient DNA extraction method from rootstock pieces of potted grapevines. Subsequently, PCR amplification using species-specific primers (Pch1 and Pch2) was found to be sensitive enough to detect as little as 1 pg of Pa. chlamydospora genomic DNA from grapevine wood. The protocol was validated using various grapevine material from 3 different rootstock cultivars (101-14 Mgt, Ramsey and Richter 99) collected from each of 3 different nurseries, including grapevines that were subjected to hot water treatment. The basal end of the rootstock was parallel analysed for Pa. chlamydospora using isolations onto artificial medium and molecular detection. The identity of PCR products obtained from a subset of samples, that only tested positive for Pa. chlamydospora based on molecular detection, was confirmed to be Pa. chlamydospora specific through restriction digestion with AatII. Molecular detection was found to be considerably more sensitive than isolations, detecting Pa. chlamydospora from samples with positive as well as negative isolations. On average, the molecular technique detected Pa. chlamydospora in 80.9% of the samples, whereas only 24.1% of the samples tested positive for Pa. chlamydospora by means of isolations. Pa. chlamydospora was not isolated from hot water treated samples. The results confirm the importance of hot water treatment for proactive management of Petri disease in grapevine nurseries. However, Pa. chlamydospora DNA was molecularly detected in hot water treated samples in frequencies similar to that detected in non-hot water treated samples. As expected, the DNA in hot water treated plants was not destroyed and could be detected by the developed molecular detection protocol. This is an important consideration when using molecular detection for disease diagnosis or pathogen detection and shows that these methods should be used in conjunction with other diagnostic tools. Most importantly, the DNA extraction protocol was shown to be 10 to 15 times cheaper than commercial DNA extraction kits. Preliminary studies showed that the aforementioned molecular detection technique was not specific and sensitive enough for detection of Pa. chlamydospora in soil and water (unpublished data). Therefore, a one-tube nested-PCR technique was optimised for detecting Pa. chlamydospora in DNA extracted from soil, water, callusing medium and grapevine wood. Rootstock cane sections and soil samples were taking from the mother blocks from several nurseries. Water samples were collected from hydration and fungicide tanks during pre-storage and grafting. Scion and rootstock cuttings were also collected during grafting and soil were collected from the nursery beds prior to planting. The one-tube nested-PCR was sensitive enough to detect as little as 1 fg of Pa. chlamydospora genomic DNA from water and 10 fg from wood, callusing medium and soil. PCR analyses of the different nursery samples revealed the presence of several putative Pa. chlamydospora specific bands (360 bp). Subsequent sequence analyses and/or restriction enzyme digestions of all 360 bp PCR bands confirmed that all bands were Pa. chlamydospora specific, except for five bands obtained from callusing media and one band from water. Considering only Pa. chlamydospora specific PCR bands, the molecular detection technique revealed the presence of Pa. chlamydospora in 25% of rootstock cane sections and 17% of the soil samples collected from mother blocks, 42% of rootstock cuttings collected during grafting, 16% of scion cuttings, 40% of water samples collected after the 12- hour pre-storage hydration period, 67% of water samples collected during grafting and 8% of the callusing medium samples. These media should therefore be considered as potential inoculum sources or infection points of the pathogen during the nursery stages. The results furthermore confirmed previous findings that Pa. chlamydospora is mainly distributed through infected rootstock canes and cuttings. Infected scion cuttings were also shown to be potential carriers of the pathogen. Management strategies should include wound protection of rootstock mother plants, eradicating this pathogen from rootstock-cuttings (e.g. hot water treatment), biological or chemical amendments in the hydration water and callusing medium and wound protection from soil borne infections.
Optimisation of postharvest drench application of fungicides on citrus fruit
(Stellenbosch : Stellenbosch University, 2016-03) Christie, Charmaine; Erasmus, A.; Fourie, P. H.; Lennox, C. L.; Stellenbosch University. Faculty of Agrisciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: South Africa is the 2nd largest exporter of fresh citrus, after Spain, worldwide. Delays to the packline, i.e. degreening, can result in substantial postharvest decay such as green mould caused by Penicillium digitatum (PD). Pre-packline aqueous fungicide drench application is an important tool to minimize postharvest losses before degreening, which provides a favourable environment for infection. Sour rot, caused by Geotrichum citri-aurantii (GC), becomes an infection risk after rainfall and the availability of effective fungicides against this pathogen is limited. Thiabendazole (TBZ), pyrimethanil (PYR), guazatine (GZT) and 2,4-dichlorophenoxyacetic acid (2,4-D) are applied during drenching in South Africa for the control of postharvest diseases on citrus, although this application has not yet been standardized and guazatine use is restricted to certain export markets; GZT is the only fungicide in the drench mixture that is effective against sour rot. Therefore the aim of this study was to improve our understanding of drench application in terms of the influence of infection age, fruit orientation (pole), treatment exposure time and the addition of adjuvants and sanitisers on disease control. Lemon, Satsuma mandarin and navel orange fruit were drenched with TBZ and PYR (1000 μg.mL-1 each) at different exposure times (14 s, 28 s and 56 s) and inoculated with PD 0, 6, 12, 18, 24, 30, 42, 48 and 54 h before (curatively) and 24 h after (protectively) treatment. Sporulation inhibition and residue loading were evaluated. Lemon and Satsuma mandarin fruit were exposed to a lower drench volume compared to navel orange fruit (26.5 and 64.3 L.min⁻¹, respectively). Batch differences played a significant role in green mould control with lemon and Satsuma mandarin fruit requiring treatment by 33.1 to 44.5 h and 23.8 to 32.1 h infection age, respectively, to gain 90% control. Exposure time only became significant with ≥ 30 h old infections on navel orange fruit at the higher drench volume used, with control declining more rapidly for fruit drenched at shorter exposure times. Control on navel orange fruit differed as much as 30.2% between exposure times with 54 h old infections and > 90% control was achieved by drenching fruit before 27 h. Protective control was generally effective (> 90%). These results support the proposition to drench all citrus types ≤ 24 h in order to reduce the risk for green mould decay development as sporulation inhibition was poor (< 50%) and fruit batches differed as much as 8 to 12 h in infection age for similar control levels. Valencia orange fruit were drenched with TBZ, PYR and 2,4-D (1000, 1000 and 250 μg.mL-1, respectively; calyx-end facing upward, sideways and downwards) at 41.0 L.min⁻¹ for 18 s with different adjuvant concentrations (0.0, 0.025, 0.05, 0.1 and 0.2 μl.mL⁻¹). Almost no differences were evident between concentrations, other than a negative effect on residue loading, deposition quantity and green mould control at the highest adjuvant concentration tested. Fruit orientation was however significant, with fruit facing calyx-end upward resulting in higher residue levels, curative green mould control, deposition quantity and quality compared to the stylar-end. Since sour rot inoculum levels can accumulate in the drench solution with dirt from fruit during drenching, Chlorine (Cl; 80 μg.mL-1) and hydrogen peroxide/peracetic acid (HPPA; 0.6%) efficacy was compared for the control of GC spores (CFU.mL-1) in solution without reducing fungicide persistence and efficacy. Wounded navel orange fruit were drenched with TBZ, PYR, GZT and 2,4-D (1000, 1000, 500 and 250 μg.mL-1, respectively) during commercial packhouse trials with Cl or HPPA (80 μg.mL-1 and 0.6%, respectively) used as shock treatments at each bin stack (two bins) containing bin no. 1, 50, 100 and 150. Fungicide persistence and green mould infection (environmental inoculum) was similar regardless of whether sanitisers were present or not. Green mould infection increased by bin 150 (4.6 – 5.4% difference). Different sanitiser concentrations (0, 20, 40, 60 and 80 μg.mL-1 Cl or 0.00, 0.01, 0.10, 0.30 and 0.60% HPPA) were combined with a mixture of TBZ, PYR and 2,4-D (1000, 1000 and 250 μg.mL-1, respectively) and GC spores (≈ 3.175 × 104 spores.mL-1) for 1, 3 and 60 min exposure during in vitro trials. Fungicide concentration was generally not influenced by sanitisers although sanitisers, however, did not persist after 60 min in solution exposed to fungicides. Only HPPA could completely reduce sour rot inoculum (0.0 CFU.mL-1) after 1 – 3 min as Cl was not as effective at the high pH levels (> 10) of the solution. During in vivo trials, green mould inoculated (24 h before treatment) and wounded fruit were drenched with TBZ, PYR and 2,4-D (1000, 1000 and 250 μg.mL-1, respectively) and GC spores (similar to in vitro trials) containing either 80 μg.mL-1 Cl or 0.3% HPPA with the addition of 0, 500 or 1000 μg.mL-1 kaolin, used to simulate dust accumulation during drenching. Sanitiser addition mostly did not affect solution concentration and green mould control, although HPPA treatments improved sour rot control on Valencia and Nadorcott mandarin fruit and resulted in improved green mould control on Nadorcott mandarin fruit; the lower level of kaolin (500 μg.mL-1) tested in this study improved green mould and sour rot control in some cases. Timeous drench application (≤ 24 h) provides effective green mould control whereas exposure time and adjuvant concentration requires further investigation in order to improve fungicide retention and distribution throughout highly congested fruit bins. Since drench pH is not regulated, HPPA was superior to Cl at high pH levels (> 10) for reducing sour rot infection and inoculum levels in solution, although further research is required to determine shock treatment intervals (within 60 min) required and potential side effects.
Pome fruit trees as alternative hosts of grapevine trunk disease pathogens
(Stellenbosch : University of Stellenbosch, 2010-03) Cloete, Mia; Mostert, Lizel; Fourie, P. H.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: A survey was undertaken on apple and pear trees in the Western Cape Province to determine the aetiology of trunk diseases with reference to trunk diseases occurring on grapevine. Grapevine trunk diseases cause the gradual decline and dieback of vines resulting in a decrease in the vine’s capability to carry and ripen fruit. In recent years, viticulture has been expanding into several of the well established pome fruit growing areas. The presence of trunk pathogens in pome fruit orchards may affect the health of the pome fruit trees as well as cause a threat to young vineyards planted in close proximity to these potential sources of viable inoculum. Several genera containing species known to be involved in trunk disease on pome fruit and grapevine were found, including Diplodia, Neofusicoccum, Eutypa, Phaeoacremonium and Phomopsis. Diplodia seriata and D. pyricolum, were isolated along with N. australe and N. vitifusiforme. Four Phaeoacremonium species, P. aleophilum, P. iranianum, P. mortoniae and P. viticola, two Phomopsis species linked to clades identified in former studies as Phomopsis sp. 1 and Phomopsis sp. 7, and Eutypa lata were found. In addition, Paraconiothyrium brasiliense and Pa. variabile, and an unidentified Pyrenochaetalike species were found. Of these the Phaeoacremonium species have not been found on pear wood and it is a first report of P. aleophilum occurring on apple. This is also a first report of the Phomopsis species and Eutypa lata found occurring on pome trees in South Africa Two new coelomycetous fungi were also found including a Diplodia species, Diplodia pyricolum sp. nov., and a new genus, Pyrenochaetoides gen. nov. with the type species, Pyrenochaetoides mali sp. nov., were described from necrotic pear and apple wood. The combined ITS and EF1-α phylogeny supported the new Diplodia species, which is closely related to D. mutila and D. africana. The new species is characterised by conidia that become pigmented and 1-septate within the pycnidium, and that are intermediate in size between the latter two Diplodia species. Phylogenetic inference of the SSU of the unknown coelomycete provided bootstrap support (100%) for a monophyletic clade unrelated to known genera, and basal to Phoma and its relatives. Morphologically the new genus is characterised by pycnidial with elongated necks that lack setae, cylindrical conidiophores that are seldomly branched at the base, and Phoma-like conidia. The phylogenetic results combined with its dissimilarity from genera allied to Phoma, lead to the conclusion that this species represents a new genus. A pathogenicity trial was undertaken to examine the role of these species on apple, pear and grapevine shoots. N. australe caused the longest lesions on grapevine shoots, while Pyrenochaetoides mali, Pa. variabile, D. seriata and P. mortoniae caused lesions that were significantly longer than the control inoculations. On pears, D. pyricolum and N. australe caused the longest lesions, followed by D. seriata and E. lata. On apples, the longest lesions were caused by N. australe and P. iranianum. D. seriata, D. pyricolum, E. lata, N. vitifusiforme, Pa. brasiliense, P. aleophilum and P. mortoniae also caused lesions on apple that were significantly longer than the control. The study demonstrated that close cultivation of grapevine to apple and pear orchards may have inherent risks in terms of the free availability of viable inoculum of trunk disease pathogens.
Singular and combined effect of postharvest treatments on viability and reproductive ability of phyllosticta citricarpa infections.
(Stellenbosch : Stellenbosch University, 2017-03) Schreuder, Wouter; Fourie, P. H.; Erasmus, A.; Du Plooy, W.; Lennox, Cheryl L.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Citrus is one of the most important fruit crops globally and is currently being produced in over 100 countries. South Africa is one of the biggest shipping exporters of fresh citrus, with approximately 40% exported to European markets. Packhouses have rigorous export quality control programmes to maintain quality and prevent postharvest decay during the shipping period. Citrus black spot (CBS) (caused by Phyllosticta citricarpa (McAlpine) van der Aa) is mostly a cosmetic disease that reduces the aesthetic quality of fruit and does not cause postharvest decay. However, P. citricarpa is regarded as a quarantine organism in certain countries, and despite scientific evidence to the contrary, trade restrictions are imposed, such as the zero tolerance for CBS lesions on fruit exported to European Union. Whilst fruit may be exported from areas where CBS occurs, very strict preharvest control programmes must be followed to ensure fruit production in orchards meet the zero tolerance requirements. The biggest danger surrounding CBS is the presence of latent, asymptomatic infections in harvested and packed fruit, which can sometimes manifest on the fruit long after packhouse treatment, cold storage and shipping. Previous studies have indicated that postharvest treatments delay symptom expression and control CBS by reducing lesion and pycnidiospore viability. The objective of this study was to evaluate the effect of more recent protocols and fungicides used in packhouses, as well as alternative fungicides, against latent CBS infections, including the reproductive potential of the lesions. Fruit with CBS lesions, as well as asymptomatic fruit with latent infections, were subjected to standard packhouse sanitation, fungicide treatment and cold storage (singularly and combined), and incubated at conditions that enable expression of latent infections. The full packhouse treatment along with storage period gave significantly control of latent infections. The over all reproductive ability of lesions were very low, with less than 2.1% of all lesion that formed on both Valencia’s and Eureka lemons developing pycnidia. Three alternative single treatments showed potential to control latent infections: FLU, potassium sorbate and Propirly 270 EC (PPZ + PYR). Treatment with (respectively) FLU and Propirly 270 EC resulted in moderate to significant control of latent infections on both Valencia oranges and Eureka lemons. Potassium sorbate moderately controlled latent CBS infections in both Valencia oranges and Eureka lemon trials. The combined epidemiological requirements for pycnidiospore release along with results from trials conducted in the current study indicate that harvested fruit is not an epidemiologically significant pathway for the spread of CBS.
The use of adjuvants to improve fungicide spray deposition on grapevine foliage
(Stellenbosch : Stellenbosch University, 2009-03) Van Zyl, Sybrand Abraham; Fourie, P. H.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Sufficient fungicide deposition on the target site is an essential requirement for effective chemical management of fruit- and foliar diseases such as grey mould of grapevines. Control failure is often attributed to insufficient quantitative deposition on susceptible grapevine tissue. However, in high disease pressure situations control failure might also be attributed to poor qualitative deposition. The primary objective of spray technology is to optimise deposition, of which the plant surface is a critical component in the spray application process, specifically in the retention of spray droplets. Adjuvant technology is reported to improve the wettability and spread of droplets by surface-acting-agents on the target surface and thereby improve deposition and retention of the fungicide active ingredient. However, this relatively new spray technology on viticulture and horticultural crops, and possible effects of adjuvants on epicuticular wax affecting plant disease development, needs to be investigated. Moreover, the development of useful prescriptions for adjuvants by determining water volumes and adjuvant dosages is required for different pesticide tank mixes. The aims of this study were, firstly to determine the effect of selected adjuvants on quantitative and qualitative spray deposition on grapevine leaves and subsequent biological efficacy of a fungicide, and secondly to evaluate selected adjuvants under field conditions and determine the effects of adjuvant dosage and spray volume on deposition. Leaves were sprayed under similar laboratory conditions to pre-run-off with 1 mL of a mixture of fenhexamid (Teldor® 500 SC, Bayer) at recommended dose, a fluorescent pigment (SARDI Fluorescent Pigment, 400 g/L EC; South Australian Research and Development Institute) at 0.2 L/100 L, as well as 15 selected commercial adjuvants to manipulate the deposition quality of a given quantity of deposited spray. Spray deposition on leaves was illuminated under black light (UV-A light in the 365 nm region) and visualised under a stereo microscope (Nikon SMZ800) at 10× magnification. Photos of sprayed leaf surfaces were taken with a digital camera (Nikon DMX 1200). Digital images were quantitatively and qualitatively analysed with Image-Pro Discovery version 6.2 for Windows (Media Cybernetics) software, to determine spray deposition. The sprayed leaves were inoculated with 5 mg dry airborne conidia of Botrytis cinerea in a spore settling tower and incubated for 24 h at high relative humidity (≥ 93%). Leaf discs were isolated onto Petri dishes with paraquat-amended water agar and rated 11 days later for development of B. cinerea from isolated leaf discs. B. cinerea incidence on the upper and lower surfaces of water sprayed leaves averaged 90.4% and 95.8%, respectively. Despite full spray cover of leaves, applications with fenhexamid alone did not completely prevent infection and resulted in 34.6% and 40.8% B. cinerea incidence on the upper and lower surfaces of leaves, respectively. Through the addition of certain adjuvants, B. cinerea incidences were significantly lower (2.9-17.1% and 10.0-30.8%, respectively), while some adjuvants did not differ from the fungicide-only treatment, even though they might have improved spray deposition. The effects of Hydrosilicote and Solitaire alone and in combination with fenhexamid on germinating Botrytis conidia on leaf surfaces were studied in a histopathology study using epifluorescence microscopy. Distinct differences were observed in conidium mortality, germination and germ tube lengths between adjuvants alone and in combination with the fungicide, which might be attributed to indirect effects of the adjuvant mode of action on B. cinerea. The laboratory study clearly demonstrated the potential of adjuvants to improve the bio-efficacy of a fungicide directly through improved deposition on grapevine leaf surfaces. For the vineyard evaluations, the same fluorometry, photomicrography and digital image analysis protocol were used to assess quantitative and qualitative spray deposits under varying adjuvant dosage and volume applications. The Furness visual droplet-rating technique was initially included to determine optimum spray volume with a STIHL SR400 motorised backpack mistblower by assessment of pigment deposition on Chardonnay leaves under illuminated black light. Both assessment protocols showed that quantitative spray deposition increased with increasing spray volume applications of 40 L/ha to 750 L/ha, but decreased at 900 L/ha, possibly due to run-off. The addition of selected adjuvants at recommended dosage and at 600 L/ha demonstrated the potential of adjuvants to increase quantitative and qualitative deposition significantly on upper and lower leaf surfaces. Agral 90, BB5, Nu-film-P, and Solitaire significantly improved deposition on upper and lower leaf surfaces compared with the fenhexamid only and water sprayed control. Break-thru S 240 and Villa 51 did not improve quantitative deposition, although remarkably better qualitative deposition was obtained. An adjuvant dosage effect (within the registered dosage range) was evident, especially those retained on the upper leaf surfaces. Agral 90 and Nu-film-P affected significant improvement of spray deposition at the higher, but not at the lower dosage tested. Solitaire improved deposition at the lower dosage tested, whereas reduced deposition at the higher dosage was attributed to excessive spray run-off. No significant improvement of spray deposition was observed for both dosages tested with Villa 51. Spray mixtures with adjuvants Agral 90 and Solitaire yielded similar deposition values at 600 L/ha compared with the fenhexamid only control at 900 L/ha, but reduced deposition at the higher spray volume, possibly due to spray run-off. This study clearly demonstrated the potential of adjuvants to improve quantitative and qualitative deposition, but highlights the necessity to match adjuvant dosages and application volumes on the spray target to achieve maximum spray deposition.

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