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Citrus tatter leaf virus (CTLV), a strain of apple stem grooving virus, is a virus of citrus that is of commercial importance for trifoliate orange (Poncirus trifoliata) and trifoliate hybrid rootstocks. Bud-union crease is a CTLV-induced incompatibly symptom observed with these rootstocks and citrus scions which results in stunted tree growth. CTLV is a regulated pathogen for most citrus budwood certification programs worldwide. Due to the requisite virus testing, commercially supplied scion budwood would therefore seldom be a point of contamination. However, citrus rootstocks are primarily propagated by seed and the potential for seed transmission of CTLV in citrus is unclear and remains a potential source of infection. A single, unconfirmed report of seed transmission of CTLV in ‘Eureka’ lemon (Citrus × limon) exists, and although this report was not of a rootstock cultivar, it does point to the risk of seed transmission in citrus and close relatives in the family Rutaceae. CTLV-positive ‘Meyer’ lemon (C. × limon) trees in an experimental plot in the Eastern Cape Province of South Africa presented an opportunity to again test seed transmission in citrus. Seed was obtained from the infected ‘Meyer’ lemon trees, and 1164 seedlings were grown in an insect-secure greenhouse and screened for CTLV in pooled batches by conventional and real-time RT-PCR. No positive samples were detected and results indicated a lack of virus seed transmission in ‘Meyer’ lemon. Findings of this study are therefore not supportive of CTLV seed transmission in citrus.

Ascosporic infection plays a major role in the epidemiology of citrus black spot (CBS) in South Africa, a disease caused by Phyllosticta citricarpa. Phyllosticta pseudothecium maturation and ascospore release models have been integrated in infection models to predict the availability of the primary inoculum source. However, these models have not been validated on a broader data set and this study aimed to validate and improve these epidemiological models. New pseudothecium maturation and ascospore release models for P. citricarpa were developed, based on weather and ascospore trap data from 13 locations and up to five seasons. From the 29 data sets analysed, 3775 3-hourly periods with ascospore events were recorded on 1798 days; 90% of these events occurred between 16.0 °C and 32.1 °C (daily Tmin and Tmax of 15.4 °C and 33.5 °C, respectively) and 75% occurred above a relative humidity (RH) of 55.9% (daily RH > 47.9%). Rain was recorded during 13.8% of these ascospore events and 20.0% of ascospore days. Using logistic regression, a Gompertz model that best predicted pseudothecium maturation, or the probability of onset of ascospore release, was developed and was markedly more accurate than the previously described models. The model consisted of DDtemp [cumulative degree-days from midwinter (1 July) calculated as (minimum + maximum daily temperature) / 2 – 10 °C] and DDwet (DDtemp accumulated only on days with >0.1 mm rain or vapour pressure deficit 0.1 mm rain or vapour pressure deficit

Huanglongbing (HLB, Asian Citrus Greening), the most devastating disease of citrus has not been detected in southern Africa (Gottwald, 2010). HLB is associated with 'Candidatus Liberibacter asiaticus' (CLas), a phloem-limited bacterium vectored by Diaphorina citri Kuwayama (Hemiptera: Liviidae), the Asian Citrus Psyllid (ACP). African Citrus Greening, associated with 'Candidatus Liberibacter africanus' (CLaf) and its vector the African Citrus Triozid, Trioza erytreae (Del Guercio) (Hemiptera: Triozidae), are endemic to Africa, although not previously reported from Angola. African Greening is less severe than HLB, largely due to heat sensitivity of CLaf and its vector. Introduction of HLB into southern Africa would be devastating to citrus production in commercial and informal sectors. Concern was raised that CLas or ACP might hae inadvertently been introduced into Angola. In July 2019, a survey was conducted in two citrus nurseries in Luanda and Caxito and in different orchards on 7 farms surrounding Calulo and Quibala. Yellow sticky traps for insects were placed at the various localities and collected after c. 3 weeks. Breeding signs of T. erytreae (pit galls) were observed on citrus in some locations, but no insect vectors were detected on traps. Trees were inspected for signs and symptoms of citrus pests and diseases, particularly those that resemble HLB (foliar blotchy mottle, shoot chlorosis, vein yellowing and corking, lopsided fruit with aborted seeds and colour inversion) and its vectors (pit galls on leaves or waxy exudates). Leaves and shoots with suspect symptoms were sampled for laboratory analysis (43 samples). DNA was extracted from petiole and midrib tissue of leaves using a modified CTAB extraction protocol of Doyle and Doyle (1990). Real-time PCR was done using universal Liberibacter primers of Roberts et al. (2015), CLaf specific primers of Li et al. (2006) and CLas specific primers of Bao et al. (2019). All real-time PCR protocols indicated the presence of CLaf in 6 samples (Tab. S1). CLas or other citrus Liberibacter species were not detected. The presence of CLaf in sample 37 was confirmed by constructing a library (NEXTFLEX® DNA Sequencing Kit, PerkinElmer) with extracted DNA and performing high-throughput sequencing on an Ion Torrent™ S5™ platform (Central Analytical Facility, Stellenbosch University). To improve the quality of the reads, all 233,617,700 obtained reads were trimmed from the 3' end to a maximum length of 240 nt using Trimmomatic (Bolger et al. 2014). The high quality reads were mapped to the Citrus sinensis reference genome (NC_023046.1) using Bowtie 2.3.4 (Langmead and Salzberg 2012) to subtract all the reads that had high identity to the host plant (number of mismatches allowed in the seed was set to 1). The 14,691,369 unmapped reads (6.2% of original data) were mapped to the CLaf reference genome NZ_CP004021.1 using CLC Genomics Workbench 10.1.1 (Qiagen) (Length fraction = 0.8; Similarity fraction = 0.9). A CLaf consensus genome was generated that spanned 99.7% of the reference genome and the 163001 mapped reads had a 22.9 mean read coverage. The consensus sequence was 99.7% identical to NZ_CP004021.1 and was submitted to Genbank as accession: CP054879. The positive CLaf detections were from trees with typical HLB or African Citrus Greening symptoms, viz. lopsided fruit with green stylar ends, aborted seed and stained columella at base of fruit button; yellow shoots with leaves showing symptoms of blotchy mottle and vein yellowing and corking (Fig. S1) in a commercial citrus farm outside Calulo and included 2 'Ponkan' mandarin (C. reticulata), 2 Valencia and 1 'Navelina' tree (C. sinensis), and a citrus nursery in Luanda (1 lime tree; C. aurantifolia) (Tab. S1). This first report of CLaf in Angola highlights the need to prevent spread by removing infected trees and managing the insect vector, as well as the need for further surveys to determine the occurrence of African Greening and its vectors in other provinces and to confirm the absence of exotic citrus pests and diseases. References Bao, M. et al. 2020. Plant Dis. 104:527 Bolger, A. M. et al. 2014. Bioinformatics. 30:2114-2120. Doyle, J.J. and Doyle, J.L. 1990. Focus 12:13 Gottwald, T.R. 2010. Annu. Rev. Phytopathol. 48:119 Langmead, B. and Salzberg, S. 2012. Nature Methods. 9:357-359. Li, W. et al. 2006. Jnl. Microbiol. Methods 66:104 Roberts, R. et al. 2015. Int. J. Syst. Evol. Micr. 65:723.

Summary Several Phyllosticta species are known as pathogens of Citrus spp., and are responsible for various disease symptoms including leaf and fruit spots. One of the most important species is P. citricarpa, which causes a foliar and fruit disease called citrus black spot. The Phyllosticta species occurring on citrus can most effectively be distinguished from P. citricarpa by means of multilocus DNA sequence data. Recent studies also demonstrated P. citricarpa to be heterothallic, and reported successful mating in the laboratory. Since the domestication of citrus, different clones of P. citricarpa have escaped Asia to other continents via trade routes, with obvious disease management consequences. This pathogen profile represents a comprehensive literature review of this pathogen and allied taxa associated with citrus, focusing on identification, distribution, genomics, epidemiology and disease management. This review also considers the knowledge emerging from seven genomes of Phyllosticta spp., demonstrating unknown aspects of these species, including their mating behaviour. Taxonomy Phyllosticta citricarpa (McAlpine) Aa, 1973. Kingdom Fungi, Phylum Ascomycota, Class Dothideomycetes, Order Botryosphaeriales, Family Phyllostictaceae, Genus Phyllosticta, Species citricarpa. Host range Confirmed on more than 12 Citrus species, Phyllosticta citricarpa has only been found on plant species in the Rutaceae. Disease symptoms P. citricarpa causes diverse symptoms such as hard spot, virulent spot, false melanose and freckle spot on fruit, and necrotic lesions on leaves and twigs. Useful websites DOE Joint Genome Institute MycoCosm portals for the Phyllosticta capitalensis (https://genome.jgi.doe.gov/Phycap1), P. citriasiana (https://genome.jgi.doe.gov/Phycit1), P. citribraziliensis (https://genome.jgi.doe.gov/Phcit1), P. citrichinaensis (https://genome.jgi.doe.gov/Phcitr1), P. citricarpa (https://genome.jgi.doe.gov/Phycitr1, https://genome.jgi.doe.gov/Phycpc1), P. paracitricarpa (https://genome.jgi.doe.gov/Phy27169) genomes. All available Phyllosticta genomes on MycoCosm can be viewed at https://genome.jgi.doe.gov/Phyllosticta.

Mancozeb spray deposition and the persistence thereof to rainfall are important factors influencing the efficacy of apple scab control caused by Venturia inaequalis. Fungicide deposition can be assessed through quantification of fungicide residue, or fluorescent pigment quantification. Fluorescent pigment quantification is a more cost effective and less labour intensive method, since it is assessed using only photomacrography and image analyses. The yellow fluorescent pigment used in this study was shown to be a suitable tracer for five different mancozeb formulations on apple leaves when evaluated at different concentrations (0.5, 1.0, 1.5 and 2.0). Pearson's correlation was significant (P 0.495) between the three treatments based on FPC% and mancozeb residue quantity when simulated rain was 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). A fair to good correlation (r = 0.697 and 0.995) existed between FPC% and mancozeb residue, and their percentage loss at different rainfall volumes. However, the response of FPC% and mancozeb residue differed based on exponential regression curves. This was due to a markedly larger predicted loss by each model's asymptote, 51.67% for FPC% and 40.76% for mancozeb residue. Based on actual mancozeb residue values, a significant percentage loss in residue already occurred after applying 1 mm (32.90%) rain. When rain volumes were increased to 5 and 10 mm rain, the percentage losses (37.88 and 41.08%) did not differ significantly from 1 mm rain. The same was true for percentage loss in FPC%, except that a significant higher loss occurred at 10 mm (52.36%) than at 1 mm rain (41.13%).

A total of 213 Penicillium digitatum isolates with resistance to the fungicide imazalil (IMZ) originating from green moulded citrus fruit from several countries (South Africa, USA, Uruguay, Spain, Israel, Cyprus, Chile, Australia and Argentina) were tested for their Cyp51 IMZ-resistance group (R1 to R3) using a published multiplex PCR assay. As well, the level of resistance (measured as EC50 values) was determined. Isolates from the USA showed the highest resistance group diversity (isolates belonged to R1, R2 and R3 groups) and lowest EC50 values. The isolates from the other countries were all classified into the R3 group, except for Chile, which also contained R1 isolates. There was no correlation between the level of resistance of isolates and the resistance group genotype. In general, IMZ resistance was most commonly associated (88.3%) with the R3 group. This study is the first to investigate the distribution of IMZ resistance genotypes in P. digitatum populations from a broad international scale.

Stress is known to perturb the microbiome and exacerbate irritable bowel syndrome (IBS) associated symptoms. Characterizing structural and functional changes in the microbiome is necessary to understand how alterations affect the biomolecular environment of the gut in IBS. Repeated water avoidance (WA) stress was used to induce IBS-like symptoms in rats. The colon-mucosa associated microbiome was characterized in 13 stressed and control animals by 16S sequencing. In silico analysis of the functional domains of microbial communities was done by inferring metagenomic profiles from 16S data. Microbial communities and functional profiles were compared between conditions. WA animals exhibited higher α-diversity and moderate divergence in community structure (β-diversity) compared with controls. Specific clades and taxa were consistently and significantly modified in the WA animals. The WA microbiome was particularly enriched in Proteobacteria and depleted in several beneficial taxa. A decreased capacity in metabolic domains, including energy- and lipid-metabolism, and an increased capacity for fatty acid and sulfur metabolism was inferred for the WA microbiome. The stressed condition favored the proliferation of a greater diversity of microbes that appear to be functionally similar, resulting in a functionally poorer microbiome with implications for epithelial health. Taxa, with known beneficial effects, were found to be depleted, which supports their relevance as therapeutic agents to restore microbial health. Microbial sulfur metabolism may form a key component of visceral nerve sensitization pathways and is therefore of interest as a target metabolic domain in microbial ecological restoration.

Objective To characterise vaginal bacterial composition in early pregnancy and investigate its relationship with first and second trimester miscarriages. Design Nested case–control study. Setting Queen Charlotte’s and Chelsea Hospital, Imperial College Healthcare NHS Trust, London. Population 161 pregnancies: 64 resulting in first trimester miscarriage, 14 in second trimester miscarriage and 83 term pregnancies. Methods Prospective profiling and comparison of vaginal bacteria composition using 16S rRNA gene‐based metataxonomics from 5 weeks’ gestation in pregnancies ending in miscarriage or uncomplicated term deliveries matched for age, gestation and body mass index. Main outcome measures Relative vaginal bacteria abundance, diversity and richness. Pregnancy outcomes defined as first or second trimester miscarriage, or uncomplicated term delivery. Results First trimester miscarriage associated with reduced prevalence of Lactobacillus spp.‐dominated vaginal microbiota classified using hierarchical clustering analysis (65.6 versus 87.7%; P = 0.005), higher alpha diversity (mean Inverse Simpson Index 2.5 [95% confidence interval 1.8–3.0] versus 1.5 [1.3–1.7], P = 0.003) and higher richness 25.1 (18.5–31.7) versus 16.7 (13.4–20), P = 0.017), compared with viable pregnancies. This was independent of vaginal bleeding and observable before first trimester miscarriage diagnosis (P = 0.015). Incomplete/complete miscarriage associated with higher proportions of Lactobacillus spp.‐depleted communities compared with missed miscarriage. Early pregnancy vaginal bacterial stability was similar between miscarriage and term pregnancies. Conclusions These findings associate the bacterial component of vaginal microbiota with first trimester miscarriage and indicate suboptimal community composition is established in early pregnancy. While further studies are required to elucidate the mechanism, vaginal bacterial composition may represent a modifiable risk factor for first trimester miscarriage. Tweetable abstract Vaginal bacterial composition in first trimester miscarriage is associated with reduced Lactobacillus spp. abundance and is independent of vaginal bleeding., Tweetable abstract Vaginal bacterial composition in first trimester miscarriage is associated with reduced Lactobacillus spp. abundance and is independent of vaginal bleeding.

Petri disease is a vascular disease associated with decline and dieback of young grapevines. A major means of spread of the causal organisms, Phaeomoniella chlamydospora and Phaeoacremonium spp., is via infected propagation material. Since no curative control measures are known, proactive measures must be taken in grapevine nurseries to manage this disease. To study this aspect, semicommercial trials with naturally infected rootstock material were performed in grapevine nurseries in South Africa. Prior to grafting, rootstocks were treated as follows: 1-h drench in suspensions of benomyl, phosphoric acid, different bacterial and Trichoderma formulations, water, or hot water treated (HWT; 30 min at 50°C). Grafted cuttings were planted and grown in a greenhouse and two commercial field nurseries and uprooted 8 months later. In instances where rootstocks were treated with benomyl or Trichoderma formulations, the incidences of Phaeomoniella and Phaeoacremonium in grafted cuttings and uprooted nursery vines were significantly lower than that of the water treatment. However, the reduction was most consistent and noteworthy in vines on rootstocks that received HWT prior to grafting. HWT of dormant nursery vines effected a similar reduction in Phaeomoniella and Phaeoacremonium incidence. Root-stock drenches in benomyl and/or Trichoderma formulations could thus be integrated with HWT for the proactive management of Petri disease in grapevine nurseries.

Imazalil (IMZ) is the most relied upon fungicide for use against citrus green mould, caused by Penicillium digitatum . In South Africa, the IMZ sulphate formulation is used as dip application in packhouses. Previous studies showed that IMZ efficacy and residue loading of this formulation is highly affected by pH in the application solution. This study investigated the effect of pH, exposure time and temperature on residue loading, green mould control and sporulation inhibition when citrus fruit were dipped in IMZ sulphate. Clementine mandarins, lemons and navel oranges were dipped in a 500 mg L −1 solution of IMZ sulphate for 15, 45 or 90 s, at a temperature of 23, 35 or 45 °C, and at pH levels of 3 or 6. At pH 3, similar residues were loaded on fruit regardless of fruit type, temperature or exposure time (1.18–1.51 mg kg −1 ). At pH 6, both temperature and exposure time influenced residue loading: at 23 °C, higher residue levels were observed than at pH 3 (2.03–2.73 mg kg −1 , increasing with longer exposure time); at 35 °C and 45 °C, residue levels increased significantly with 15 s, 30 s and 45 s exposure time (3.34, 5.11, 7.24 mg kg −1 and 5.31, 9.65 and 13.10 mg kg −1 , respectively). Clementine mandarin fruit loaded higher residues at pH 6 (6.96 mg kg −1 ) than lemons and navels (5.20 and 4.82 mg kg −1 , respectively). The MRL was frequently exceeded at longer exposure times. Green mould control was lower at pH 3 and was influenced by temperature and exposure time (92.9–97.4% and 42.8–45.1% for lemons and Clementine mandarins, respectively). Better control was observed at pH 6 regardless of temperature and exposure time (96.9–98.7% and 39.3–57.9% for lemons and Clementine mandarins, respectively). On navel oranges, increased exposure time led to increased green mould control (84.7–92.5%). Sporulation incidence was lower at pH 6 (12.5–28.8%, 19.1–39.2% and 4.6% for lemons, Clementine mandarins and navel oranges, respectively) than at pH 3 treatments (72.7–89.9%, 35.8–91.4% and 77.2% for lemons, Clementine mandarins and navel oranges, respectively). The results show that temperature, pH and exposure time are important parameters in dip application using IMZ sulphate formulation with significant effects on green mould control, more so than residue loading alone.

Citrus Research International, Citrus Academy, Postharvest Innovation Fund (PHI) and Technology and Human Resources for Industry Programme (THRIP).

Male olive (Papio anubis) and hamadryas (P. hamadryas) baboons have distinctive sociobehavioral and physical characteristics. In the Awash National Park, Ethiopia, a hybrid population at the contact zone between these two species, exhibits heterogeneous sociobehavioral and physical characteristics. The ambiguity of the hybrid social environment and disruption of parental stress genotypes may be sources of physiological stress for hybrids. We examined levels of chronic stress among males of the three populations and tested the prediction that chronic cortisol levels would be higher among the hybrids. Animals were captured, sampled, and released during the wet season, and a hair sample was taken for assay. Cortisol was extracted from 182 hair samples with methanol and quantified by ELISA. We included age, age class, rainfall variation, and species affiliation in models examining variation in hair cortisol levels. Species and age significantly contributed to models explaining variation in hair cortisol. Infant hypercortisolism was observed in all three groups, and a decline in cortisol through juvenile and adolescent stages, with a subsequent rise in adulthood. This rise occurred earliest in hamadryas, corroborating other evidence of the precocious development of hamadryas baboons. As expected, hybrids had significantly elevated hair cortisol compared with olive baboons and hamadryas, irrespective of age, except for very young animals. Infant hypercortisolism was also less pronounced among hybrids. Species differences and age-related differences in cortisol levels suggest a dysregulated cortisol phenotype in hybrids, and possibly reflect some form of hybrid disadvantage. More work will be required to disentangle the effects of genetic factors and the social environment.

Green mould ( Penicillium digitatum) is a major cause of postharvest losses in citrus. Wounds and infection can be inflicted during the harvest process and should be controlled during postharvest stages to prevent decay. Imazalil (IMZ) in the sulphate formulation is currently applied by the majority of South African packhouses through an aqueous dip treatment. In this study the effects of incubation time (infection age), exposure time, solution pH, wounds size and fruit brushing after dip treatments on residue loading and curative green mould control were investigated. Exposure time did not have a significant effect on residue loading on fruit dipped in pH 3 solutions of IMZ ( −1 ). Increasing the pH to 6 resulted in significantly increased residue loading, which increased with longer exposure time, but mostly to levels below the maximum residue level of 5 μg g −1 after 180 s. Post-dip treatment brushing reduced residue levels obtained in IMZ pH 3 solutions by up to 90% to levels −1 ; however, curative control of the IMZ sensitive (S) isolate was mostly unaffected, but with poor sporulation inhibition. At pH 6, post-dip brushing reduced residues to ≈60%; again curative control of the sensitive isolate was unaffected, but with better sporulation inhibition than the pH 3 treatments. Wounded rind sections loaded higher residue levels compared with intact rind sections, and large wounds loaded higher levels than small wounds (≈10.19, ≈9.06 and ≈7.91 μg g −1 for large, small and no wound, respectively). Curative control of infections originating from large wounds was significantly better than those from small wounds. The ability of IMZ to control sensitive green mould infections declined from 6 and 12 h after inoculation on Clementine mandarin fruit of infections inflicted by small and large wounds, respectively; on navel orange fruit, curative control declined 18 and 36 h after inoculation for the respective wound size treatments. This work shows the importance of timely fungicide treatment after harvest especially on more susceptible citrus types. Results also indicate that excess residues can be stripped from the fruit, retaining residues necessary for curative control in the wound sites. However, reduced residue loading compromised the sporulation inhibition activity of IMZ.

The genus Phyllosticta occurs worldwide, and contains numerous plant pathogenic, endophytic and saprobic species. Phyllosticta citricarpa is the causal agent of Citrus Black Spot disease (CBS), affecting fruits and leaves of several citrus hosts (Rutaceae), and can also be isolated from asymptomatic citrus tissues. Citrus Black Spot occurs in citrus-growing regions with warm summer rainfall climates, but is absent in countries of the European Union (EU). Phyllosticta capitalensis is morphologically similar to P. citricarpa, but is a non-pathogenic endophyte, commonly isolated from citrus leaves and fruits and a wide range of other hosts, and is known to occur in Europe. To determine which Phyllosticta spp. occur within citrus growing regions of EU countries, several surveys were conducted (2015–2017) in the major citrus production areas of Greece, Italy, Malta, Portugal and Spain to collect both living plant material and leaf litter in commercial nurseries, orchards, gardens, backyards and plant collections. A total of 64 Phyllosticta isolates were obtained from citrus in Europe, of which 52 were included in a multi-locus (ITS, actA, tef1, gapdh, LSU and rpb2 genes) DNA dataset. Two isolates from Florida (USA), three isolates from China, and several reference strains from Australia, South Africa and South America were included in the overall 99 isolate dataset. Based on the data obtained, two known species were identified, namely P. capitalensis (from asymptomatic living leaves of Citrus spp.) in Greece, Italy, Malta, Portugal and Spain, and P. citricarpa (from leaf litter of C. sinensis and C. limon) in Italy, Malta and Portugal. Moreover, two new species were described, namely P. paracapitalensis (from asymptomatic living leaves of Citrus spp.) in Italy and Spain, and P. paracitricarpa (from leaf litter of C. limon) in Greece. On a genotypic level, isolates of P. citricarpa populations from Italy and Malta (MAT1-2-1) represented a single clone, and those from Portugal (MAT1-1-1) another. Isolates of P. citricarpa and P. paracitricarpa were able to induce atypical lesions (necrosis) in artificially inoculated mature sweet orange fruit, while P. capitalensis and P. paracapitalensis induced no lesions. The Phyllosticta species recovered were not found to be widespread, and were not associated with disease symptoms, indicating that the fungi persisted over time, but did not cause disease.

The sensitivity to folpet of Botrytis cinerea isolates obtained from table grape vineyards in the Western Cape province of South Africa with a known history of dicarboximide (DC) resistance and high-schedule DC and folpet programmes was investigated. In the Simondium vineyards, 61 % of the B. cinerea isolates from Dan-hen-Hannah and 20% of the isolates from Waltham Cross were resistant to iprodione. In the Northern Paarl vineyards, 95% of the isolates from Dan-hen-Hannah and 95% of the isolates from Waltham Cross were designated resistant. In the case of the iprodione-sensitive isolates from vineyards in Simondium, folpet ECso values ranged from 4.9 to 29.1 μg/mL for the Dan-hen-Hannah and 15.0 to 43.5 μg/mL for the Waltham Cross sub-populations, respectively. Folpet ECso values of the iprodione-resistant isolates, on the other hand, ranged from 19.7 to above 100 μg/mL for the Dan-hen-Hannah subpopulation. In the Northern Paarl subpopulations, where the isolates were predominantly iprodione-resistant, folpet ECso values of the latter isolates ranged from 21.5 to above 100 μg/mL. Similar shifts in folpet sensitivity were displayed by ultra-low- and low-level DC-resistant B. cinerea isolates obtained from other regional subpopulations. The results indicated incomplete cross-resistance between iprodione and folpet. This finding suggests that early increases in DC resistance frequencies in B. cinerea, observed prior to DC application in vineyards under the high-schedule DC and folpet programmes, can be attributed to incomplete cross-resistance to these fungicides in sub-populations of the pathogen.

Seasonal fluctuations in the frequency of dicarboximide-resistant Botrytis cinerea isolates in Western Cape vineyards suggest a reduced fitness of these isolates. In this study fitness of sensitive, ultra-low-level and low-level dicarboximide-resistant isolates of B. cinerea were compared on grape berries. Conidia were dispersed as single cells on berry surfaces from airborne inoculum in a settling tower or deposited as clusters in water droplets. Investigations were conducted at two potential infection courts on dicarboximide treated and untreated berries, namely the intact cuticle and wounds. Surface sterilisation, isolation and freezing techniques were used to determine surface colonisation and penetration by airborne inoculum. Decay incidence, severity and sporulation incidence were determined following inoculation with conidial clusters. The different tests indicated that germlings of dicarboximide-sensitive and -resistant isolates had similar surface-colonising abilities of dicarboximide-free berries. However, sensitive strains penetrated significantly more often. Fitness decreased with an increase in the level of dicarboximide resistance. Iprodione caused a drastic disturbance in the ratio of different dicarboximide sensitivity classes that occupied the berry surface and allowed the development of germlings of predominantly resistant isolates, but with few successful infections. Significantly higher levels of infection and proliferation of dicarboximide-resistant isolates on sprayed or unsprayed berries were facilitated by wounding or the termination of host resistance (freezing). According to these findings, these modes of infection should not contribute to a gradual build-up of inoculum of either dicarboximide-sensitive or -resistant isolates. Trends by airborne conidia described here suggest that another primary infection event in the vineyard, most likely floral infection and subsequent debris colonisation, should largely regulate the dynamics between dicarboximide-sensitive and -resistant isolates in B. cinerea populations on grapevine.

Between 1993 and 1995, 1139 isolates of Botrytis cinerea were collected from table grape vineyards in the Western and Northern Cape provinces and tested for sensitivity to dicarboximides (vinclozolin) and benzimidazoles (carbendazim). Overall the frequency of resistance to vinclozolin and carbendazim averaged 9,4 and 11,9%, respectively. Ninety-seven percent of the vinclozolin-resistant isolates were dual resistant to carbendazim. Fifty-nine percent of the isolates were from the Hexriver region; of these 7,7% were vinclozolin, and 12,6% carbendazim resistant. The Paarl region had the highest resistance incidence (37,7% vinclozolin and 41,5% carbendazim resistant). Only two of the 191 isolates tested from the Orange River region were vinclozolin resistant. ED50 values of ultra-low-level and low-level dicarboximide-resistant isolates ranged from 0,843 to 1,834 and 2,053 to 5,129 μg vinclozolin/ml, respectively. Vinclozolin-resistant strains were not abnormally osmotically sensitive. Monitoring of changes in the frequency of resistance in 10 commercial vineyards under high-, medium- and low-schedule dicarboximide programmes from 1993-1995 showed that the resistance frequency fluctuated from low (average 12,7% ) during the winter to high (average 55,8 % ) during the growing season. Maximum levels of dicarboximide resistance were recorded during bunch closure. Distinct differences were, however, observed in the resistance frequency in individual vineyards. In four high-schedule dicarboximide vineyards resistance frequencies increased early in the season, prior to any dicarboximide applications. This finding suggests dual resistance between broad-spectrum fungicides and dicarboximides.

Grapevine downy mildew, caused by Plasmopara viticola, is a sporadic, but destructive disease of grapevines in South Africa. Despite extensive fungicide spray programmes, disease control during disease-conducive weather periods is often inadequate, and severe losses were experienced during recent epidemics. Poor disease control can be attributed to several factors, one of which may be reduced efficacy of fungicides due to resistance build-up in the pathogen populations. P. viticola isolates were sampled during the 1999/2000, 2000/2001 and 2001/2002 seasons from various vineyards in the Western Cape province of South Africa and screened for sensitivity to metalaxyl using the floating leaf-disc method recommended by the Fungicide Resistance Action Committee. Minimum inhibitory concentration (MIC) values for 813 isolates were determined. Only 5.8% of the isolates tested had MIC values of 10 μg/mL and lower. The majority of the isolates (94.2 % ) showed MIC values of 100 μg/mL or more. These isolates were designated resistant to highly resistant. With the exception of one vineyard in a remote area where phenylamides had not previously been used, very high resistance frequencies were observed, irrespective of whether the samples were taken early or late in the season. This observation, supported by the consistent high resistance frequencies recorded over time in individual vineyards, is indicative of the stability of metalaxyl resistance in grapevine downy mildew populations. In view of the widespread occurrence and stability of resistance against metalaxyl, the use of phenylamide formulations for the control of downy mildew in Western Cape vineyards should be reconsidered.

The effect of fungicide spray cover on Botrytis cinerea infection in grape bunches was determined. Bunches were sprayed at pea size and bunch closure with different volumes of a mixture of fenhexamid and a yellow fluorescent pigment, and the percentage fluorescent pigment coverage on pedicels was determined. Bunches were subsequently dusted with dry airborne conidia of B. cinerea in a settling tower and incubated for 24 h at high relative humidity (98%). Infection was determined by estimating the amount of B. cinerea infections on susceptible bunch parts (pedicel, receptacle and rachis) with isolations onto paraquat (herbicide) and Kerssies (B. cinerea selective) mediums. Linear regressions for the part × stage combinations of percentage B. cinerea incidence on different bunch parts were fitted on mean coverage levels. An increase in spray cover caused linear reductions in levels of B. cinerea on bunch parts. Higher B. cinerea incidences were recorded at pea size. Furthermore, higher B. cinerea incidences were found on paraquat medium for both stages, than on Kerssies medium. The information gathered from this study will be used to facilitate future determination of minimum effective coverage levels for effective B. cinerea control in grape bunches.

Green mould (caused by Penicillium digitatum) is a major cause of postharvest losses in citrus. Residue loading of thiabendazole (TBZ) with application methods typically used in South African packhouses and green mould control was studied. TBZ was applied curatively and protectively in dip, drench and wax coating treatments and fruit were inoculated with a TBZ-sensitive or a TBZ-resistant isolate of P. digitatum. The dip treatments consisted of TBZ concentrations of 0–2000 μg mL−1; fruit were dipped for 60 s at 22 °C at a pH of 7. Residues differed between fruit batches and ranged from 0.5 to 1.7 μg g−1 at 1000 μg mL−1 TBZ. Curative dip treatments almost completely controlled green mould (>96% at 1000 μg mL−1 TBZ). The residue level needed for 75% curative control ranged from 0.06 to 0.22 μg g−1, depending on citrus type. Protective treatments were unreliable and control varied from 17% to 97.9% at 1000 μg mL−1 TBZ between fruit batches. Drench treatments consisted of exposure times of 30, 60 and 90 s with 1000 or 2000 μg mL−1 TBZ. Average TBZ residues were 2.14 μg g−1 for Clementine mandarin fruit and 3.50 μg g−1 for navel orange fruit. Green mould control on navel orange fruit resulted in 66–92%, 34–90% and 9–38% control for curative treatments after 6 and 24 h and protective treatments, respectively, depending on fruit batch. Wax with 4000 μg mL−1 TBZ was applied at 0.6, 1.2 and 1.8 L wax ton−1 fruit. Chilling injury was evaluated after fruit storage at −0.5 °C for 40 days. Average TBZ residues loaded was 1.3, 1.3 and 2.7 μg g−1 at the recommended 1.2 L ton−1 for Satsuma mandarin, Clementine mandarin and Valencia orange fruit, respectively. Protective treatments showed lower infection levels (14–20%) than curative treatments (27–40%) for Valencia orange fruit. The same trend was observed with Satsuma (92–95% curative; 87–90% protective) and Clementine mandarin fruit (82–90% curative; 59–88% protective), but control was relatively poor. TBZ application in wax exceeded 5 μg g−1 at higher wax loads (1.2 and 1.8 L ton−1). Wax treatments showed a significant reduction in chilling injury; TBZ had an additive effect. TBZ resistant isolates could not be controlled.

Eutypa dieback, caused by the fungus Eutypa lata, is a serious disease of grapevines that infects mainly through pruning wounds. The aim of this study was to evaluate the in vitro efficacy of fungicides from various chemical groups against E. lata, as well as the in vivo efficacy of the most effective fungicides and selected bacterial and fungal antagonists of E. lata, in grapevine pruning wound protection trials. In vitro studies revealed that flusilazole, tebuconazole, benomyl, fenarimol and myclobutanil were the most effective fungicides to inhibit mycelial growth of E. lata. Two field trials were conducted, one subjected to artificial inoculation and the second to natural infection only. In the first, benomyl, flusilazole and commercially available Trichoderma harzianum-containing products and an experimental Bacillus subtilis strain were applied to fresh pruning wounds. Two Cabernet Sauvignon vineyards were pruned in August 2001 and 2002 and immediately treated and inoculated with a spore suspension of E. lata one day later. Isolations were made from the treated pruning wounds after 12 months to assess the effectiveness of the treatments. The fungicides benomyl and flusilazole were the most effective treatments, although the Trichoderma treatments T77 and Trichoseal spray caused a significant reduction in E. lata infection. In a second trial, pruning wounds of Cabernet Sauvignon, Sauvignon blanc, Red Globe and Bonheur were treated with the Trichoderma products Vinevax (= Trichoseal spray) and Eco77 (= T77) in August 2005 and 2006, subjected to natural infection only and evaluated after seven months. Vinevax and Eco77 not only reduced E. lata, but they also reduced the incidence of other grapevine trunk disease pathogens.

Wax application plays an important role in prolonging fruit quality, and the addition of imazalil (IMZ) furthermore protects fruit against green mould caused by Penicillium digitatum. The objectives of this study were to evaluate green mould control and quality preservation effects of carnauba or polyethylene citrus coatings supplemented with IMZ, as well as the effect of synthetic or horsehair brush types used on sweet orange fruit. Single applications of IMZ at 3000 μg mL −1 at rates of 0.6, 1.2 and 1.8 L t −1 resulted in residues that increased with increasing coating loads on navel oranges (1.31 to 3.32 μg g −1 ) and Valencia oranges (3.22 to 6.00 μg g −1 ). Coating with IMZ generally provided poorer curative control (≈14%) than protective control (≈58%), with less sporulation in treatments using horsehair (≈59%) than synthetic brushes (≈64%). More fruit weight and firmness losses were found in fruit treated with the polyethylene coating (≈1.18 and ≈0.93 ratios of treated vs . untreated, respectively) and lower in carnauba treated fruit (≈0.76 and ≈0.74 ratios, respectively). However, polyethylene coatings resulted in shinier fruit before (≈10.85 shine ratio) and after storage (11.60), whereas carnauba coatings resulted in lower shine ratios (≈7.45 and 10.15, respectively). Gas (CO 2 ) exchange ratios remained similar for both waxes (≈0.67). Higher polyethylene coating loads (1.8 L t −1 ) resulted in off-tastes similar to uncoated control fruit (≈2.21 rating on a 5-point scale) and higher than the rating for carnauba coated fruit (≈1.82) at this rate. Scanning electron micrographs showed an amorphous crystallised natural wax layer with uncovered stomatal pores on the surface of uncoated fruit. The thickness of the applied wax layer increased with increasing coating load. A single application of IMZ in wax provided good protective green mould control and sporulation inhibition, with differing effects on some fruit quality parameters due to coating and brush types.

Inadequate disease control on citrus foliage and fruit is often attributed to insufficient fungicide spray deposition on target surfaces. This study describes a novel spray deposition assessment protocol and determines deposition benchmarks indicative of the biological effectiveness for better interpretation of spray deposition results. Suitability of a yellow fluorescent pigment as tracer for copper oxychloride deposition was demonstrated through its similar particle concentration and size. Spray deposition assessment of spray targets, which were sprayed with a mixture that included the fluorescent pigment, involved photomacrography of whole leaf or fruit surfaces, followed by digital image analyses. This protocol proved to be very accurate in determining the quantity and quality of deposition. To determine deposition benchmarks, detached young ‘Nova’ mandarin leaves were sprayed with copper oxychloride and fluorescent pigment at different concentrations (0.1–2 times the recommended concentration) and spray deposition assessed. Subsequently, leaves were spray inoculated with a spore suspension of Alternaria alternata [causal agent of Alternaria brown spot (ABS) of mandarins], moist-incubated for c. 48 h and symptom expression rated. A very good linear relationship was found between fungicide concentration, leaf area covered by fluorescent pigment particles (%FPC) (r = 0.879) and Cu residue analysis (r = 0.992). A von Bertalanffy growth curve best fitted the relation between ABS control and deposition quantity (%FPC) data (91% of the percentage variance accounted for) with a good correlation between observed and predicted values (r = 0.825). Benchmarks for 50% and 75% disease control were calculated as 2.07 %FPC and 4.14 %FPC, respectively. These corresponded with Cu residue levels of 59.4 and 91.0 mg kg−1, respectively. These FPC benchmarks can be used to evaluate spray technology research, specifically for control of ABS and similar citrus fruit and foliar diseases.

Ascospores are the most important inoculum source of citrus black spot (CBS), caused by Guignardia citricarpa, but pseudothecium maturation and ascospore release are inadequately studied. Guignardia ascospore trapping and concomitant weather data were obtained for three localities over three seasons (July to March 2006 to 2009) in the Limpopo province of South Africa. Degree-days accumulated until first seasonal ascospore discharge (>10°C with 1 July as biofix) (DDtemp), and DDtemp accumulated on rainy (rainfall >0.1 mm) (DDrain) and moist days (vapor pressure deficit 2 = 0.608 and 0.658, respectively). Both models predicted a delay in pseudothecium maturation in climates with colder winters and springs. A Gompertz equation was also used to predict the proportion of Guignardia ascospores trapped (PAT) per season from DDtemp data accumulated on wet or moist days (DDwet2) from the first seasonal ascospore discharge [PAT = exp(–4.096 × exp(–0.005 × DDwet2); R2 = 0.908]. The PAT model predicted lag phases and 7-day peaks in ascospore release patterns with reasonable accuracy. These models can be used to predict the onset and dynamics of ascospore release in climatically diverse regions.

Imazalil (IMZ) is widely used in citrus packhouses to manage green mould, caused by Penicillium digitatum . The aim of this study was to investigate green mould control efficacy of IMZ applied in a wax coating, and the combination of aqueous dip and coating IMZ applications. Single application of IMZ at 3000 μg mL −1 in carnauba wax coating at rates of 0.6, 1.2 and 1.8 L tonne −1 of fruit gave better protective (mean 13% infection) than curative (mean 70% infection) control of the sensitive isolate. Imazalil residue levels increased (0.85 to 1.75 μg g −1 ) with increasing coating load. However, the resistant isolate could not be controlled (>74% infection). Dip only treatment (IMZ sulphate at 500 μg mL −1 for 45 s and 90 s) gave good curative control (≈77%) of the sensitive isolate at residue loading of 0.12–0.73 μg g −1 . Wax coating only treatment (IMZ at 3000 μg mL −1 at 1.8 L wax tonne −1 ) gave good protective control and improved sporulation inhibition (≈80%) at residue loading of 1.32–7.09 μg g −1 . The MRL of 5 μg g −1 was exceeded at higher wax loads on navels and clementines. Double application with dip (45 s in IMZ sulphate at 500 μg mL −1 ) followed by 2000 μg mL −1 IMZ in wax coating at 0.6, 1.2 and 1.8 L wax tonne −1 resulted in residue loading of 1.42 to 2.83 μg g −1 , increased protective control (≈69%) as well as curative control (≈83%). In all treatments, poor curative and protective control of the resistant isolate was observed (

Green mould, caused by Penicillium digitatum, is responsible for major postharvest fruit losses on the South African fresh citrus export market. Some of these losses as well as fungicide resistance development can be attributed to sub-optimal imazalil (IMZ) residue loading on citrus fruit (

Botrytis cinerea causes severe losses of rooibos seedlings in the Clanwilliam region of the Western Cape of South Africa. A total of 207 isolates were collected from diseased seedlings representing five nurseries. The cryptic species status of these isolates was determined with restriction enzyme analysis of the Bc-hch gene with 206 of the isolates identified as B. cinerea and one isolate as B. pseudocinerea. Analysis of the B. cinerea populations using seven microsatellite loci revealed a high total gene diversity (H), with a mean of 0.67. Analysis of molecular variance indicated that 97 % of the total genetic variation is distributed within subpopulations. Genotype flow was evident between nurseries indicated by shared occurrence of clonal lineages, in agreement with the relatively low but significant population differentiation between nurseries (mean F ST = 0.030, P = 0.001). The distribution of the mating type alleles MAT1-1 and MAT1-2 differed significantly from the ratio of 1:1 for the total population and in two of the five nursery subpopulations. Together with significant linkage disequilibrium found, populations appear to mainly reproduce asexually. Fungicide resistance frequency against iprodione for 198 of the genotyped isolates, showed highly varying levels of resistance between the nurseries. The mean incidence of resistance towards iprodione was 44 % in total, ranging from 0 % to 81 % for the five nurseries. This study has shown an adaptive capacity to overcome current chemical means of control in the studied B. cinerea population.

High volumes of copper hydroxide, cuprous oxide and copper oxychloride were sprayed under natural conditions onto mature orange trees to compare the retention on citrus leaves and fruit over a period of 56 days by means of copper residue analyses and a spray deposition assessment protocol using fluorometry, photomacrography and digital image analyses. Rainfall and increase in fruit size were also recorded to determine if it had an influence on weathering of copper residues. Initial retention following application of the different fungicides differed on Valencia leaves and fruit: applications with cuprous oxide retained significantly more copper residue and fluorescent pigment, while copper hydroxide retained higher copper and pigment levels on Navel leaves and fruit. Nonetheless, persistence of copper residues deposited by the three copper fungicide formulations was similar and decreased at the same tempo during both seasons; initially a fast reduction (48 and 60% for year one and two respectively) in residue during the first 14 days followed by a more gradual decline (41 and 24% for year one and two respectively) from 14 to 56 days. The loss of copper residues was attributed to weathering (days after treatment), fruit growth and cumulative rainfall as these factors were inversely correlated with copper residue levels (Pearson's r = −0.840, −0.722 and −0.733 respectively). A 76% and 90% correlation was observed between the copper residue analyzed and the quantitative fluorescent pigment measurements on mature leaves and fruit, respectively; showing that fruit is more reliable for fluorometry analyses and that this technique proved to be an effective tool for spray deposition and persistence assessment of copper fungicides. All copper formulations tested at these registered rates at 35-day spray intervals were effective in controlling Guignardia citricarpa .

Insufficient quantity but also quality of spray deposition on susceptible grapevine tissue (i.e. target sites) and favourable conditions for pathogens could lead to control failure during high disease pressure situations. To determine deposition quantity and quality benchmarks for biologically effective spray deposits, bunches and leaves of table grapes (Waltham Cross) were sprayed at various growth stages, using different application volumes of a mixture of fenhexamid and a fluorescent tracer pigment and subsequently dusted with dry conidia of Botrytis cinerea where after infection levels on pedicels, receptacles and leaves were determined. Pigment deposition quantity and quality were determined from photos of sprayed parts taken with a digital camera under a stereo microscope and black light illumination at ×30 or ×10 magnifications and assessed with digital image and Hoerl regression analyses. The deposition quantity resulting in 75% control of B. cinerea infection (FPC75 values) was calculated from biological efficacy curves (sigmoidal regression analyses) for leaves and for each growth stage, for pedicels and receptacles. Deposition quantity and quality measurements correlated favourably with Botrytis infection. An optimal deposition value for the control of B. cinerea was determined by increasing spray volume, however by increasing spray volume and deposition quantity or quality levels past this optimum will not significantly improve disease control further. It was indicated that efficacy of agricultural chemicals could be influenced by improving both deposition quantity and quality, quantifiable by digital image analyses of fluorescent pigment deposition. FPC75 values obtained in this study can be used as benchmarks to evaluate future spray application in vineyards.

The aim of this study was to compare 11 treatment regimes (TR) in grapevine nurseries in order to eradicate trunk pathogens. Grapevine propagation material was subjected to treatments before cold storage, before and after grafting, before planting and after uprooting. Isolations were made from roots, rootstocks and graft unions of uprooted vines. None of the treatments consistently affected the number of certifiable vines produced. TR 1 (benomyl), TR 3 (Sporekill) and TR 4 (Trichoflow) increased root mass. Phaeomoniella chlamydospora was the most frequently isolated pathogen from all plant parts. In general, TR 2, 7, 9 and 11 caused the lowest occurrence of Phaeomoniella. TR 9 consistently reduced the incidence of Phaeoacremonium. Pleurostomophora richardsiae occurred most frequently in graft unions, but treatments were too inconsistent to draw meaningful conclusions. TR 7 (hot water treatment (HWT) of uprooted grapevines) consistently reduced incidences of black foot disease (BFD) pathogens. The highest incidence of Botryosphaeriaceae occurred in graft unions, but all treatments significantly reduced infections. The colonisation of rootstocks and graft unions by Trichoderma was significantly better with TR 4 (Trichoflow). The other Trichoderma treatments (TR 9, 10 and 11) differed from the control treatment only in the graft unions. This is a first report of an integrated strategy covering all the phases of the propagation process, from the moment the nursery receives the propagation material until the dormant vines are removed. TR 9 is recommended for use in nurseries, although HWT of dormant vines is recommended to eradicate BFD pathogens. Benomyl can be replaced by carbendazim when benomyl is no longer available.

Imazalil (IMZ) is commonly applied in South African citrus packhouses for the control of green mould, caused by Penicillium digitatum, yet the disease still causes significant postharvest losses. The maximum residue limit (MRL) for IMZ on citrus fruit is 5 μg g−1, whereas 2–3 μg g−1 is a biologically effective residue level that should at least inhibit green mould sporulation. Standard compliance auditing of residue levels of citrus fruit, however, indicate that fruit from the majority of packhouses have residues of ≈1 μg g−1. Poor disease control from insufficient residue loading might further be compounded by the presence of IMZ-resistant isolates of P. digitatum in packhouses. This study was conducted to assess the current status of IMZ application in South African packhouses, to determine the adequate residue levels needed to control green mould and inhibit its sporulation using both IMZ sensitive and resistant isolates, to investigate IMZ application methods and resultant residue levels in commercial citrus packhouses, and to study optimisation of modes of IMZ application in citrus packhouses. Factors studied were IMZ concentration, application type (spray vs. dip and drench), exposure time, solution temperature and pH, as well as curative and protective control of P. digitatum. The packhouse survey showed that the majority of packhouses applied IMZ in a sulphate salt formulation through a fungicide dip tank, and loaded an IMZ residue of ≈1 μg g−1. In dip applications, IMZ had excellent curative and protective activity against Penicillium isolates sensitive to IMZ. However, curative control of IMZ resistant isolates was substantially reduced and protective control was lost, even at twice the recommended concentration, nor was sporulation inhibited. The use of sodium bicarbonate (2%) buffered imazalil sulphate solutions at pH ±8, compared with pH ±3 of the unbuffered solutions, markedly increased IMZ residue loading on Navel and Valencia oranges and improved curative and protective control of IMZ resistant isolates. Exposure time did not affect IMZ residue loading in IMZ sulphate solutions at pH 3, although the MRL was exceeded after 45 s exposure in pH 8 solutions. Imazalil applied through spray or drench application improved residue loading, but green mould control was less effective than after dip application.

Species of the genus Coniochaeta (anamorph: Lecythophora) are known as pathogens of woody hosts, but can also cause opportunistic human infections. Several fungi with conidial stages resembling Lecythophora were isolated from necrotic wood samples of Prunus trees in South Africa. In order to reveal their phylogenetic relationships, these fungi were studied on a morphological and molecular (5.8S nrDNA, ITS-1, ITS-2, GAPDH, EF-1α, 28S nrDNA, 18S nrDNA) basis. Some of the isolates were identified as Coniochaeta (Sordariomycetes), including C. velutina and two new species, C. africana and C. prunicola. The majority of the isolates, however, formed pycnidial or pseudopycnidial synanamorphs and were not closely related to Coniochaeta. According to their 28S nrDNA phylogeny, they formed two distinct groups, one of which was closely related to Helotiales (Leotiomycetes). The new genus Collophora is proposed, comprising five species that frequently occur in necrotic peach and nectarine wood, namely Co. africana, Co. capensis, Co. paarla, Co. pallida and Co. rubra. The second group was closely related to Phaeomoniella chlamydospora (Eurotiomycetes), occurring mainly in plum wood. Besides P. zymoides occurring on Prunus salicina, four new species are described, namely P. dura, P. effusa, P. prunicola and P. tardicola. In a preliminary inoculation study, pathogenicity was confirmed for some of the new species on apricot, peach or plum wood.

Trunk disease pathogens of grapevines, viz. Phaeomoniella chlamydospora, Eutypa lata and several species in Botryosphaeriaceae, Phaeoacremonium and Phomopsis are known to infect fresh pruning wounds by means of air-borne inoculum released after rainfall or prolonged periods of high relative humidity. Recent surveys have demonstrated that most or all of these pathogens are present in climatically diverse grape growing regions of South Africa. However, the factors controlling spore dispersal of these pathogens in vineyards were largely unknown. To address this question, spore trapping was done in a Chenin Blanc vineyard in the Stellenbosch area, South Africa, for 14 weeks during the grapevine pruning period from June to mid-September of 2004 and 2005. Hourly recordings of weather data were done by a weather station in the row adjacent to the spore trap. Spores of E. lata and Phomopsis and species in Botryosphaeriaceae were trapped throughout the trapping periods of 2004 and 2005, with higher levels of trapped spores recorded in 2005. The spores of all three pathogens were trapped during or after periods of rainfall and/or high relative humidity. In neither of the 2 years were spores of Pa. chlamydospora or Phaeoacremonium spp. trapped. Results indicated that spore event incidence, as well as the amount of spores released during a spore event of above-mentioned pathogens, were governed by rainfall, relative humidity, temperature and wind speed prior to and during the spore events.

Spray adjuvants have the potential to improve deposition by effecting uniform distribution of the active ingredient on plant surfaces. In order to study whether such a qualitative improvement of spray deposition would lead to improved disease control, a laboratory experiment was conducted on artificially inoculated grapevine (cv. Chardonnay) leaves. Prior to inoculation with Botrytis cinerea conidia in a spore settling tower, leaves were sprayed to pre-runoff with 1 mL of a mixture of fenhexamid, a fluorescent pigment, and one of 15 selected commercial adjuvants to manipulate the deposition quality of a specific quantity of spray. Following an incubation period of 24 h at high relative humidity, leaf discs were plated onto Petri dishes with paraquat-amended water agar and rated for development of B. cinerea from isolated leaf discs 11 d later. Spray deposition on leaves was assessed with a spray assessment protocol using fluorometry, photomicrography and digital image analyses. B. cinerea incidences 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 incidences on the upper and lower surfaces of leaves, respectively. Through the addition of certain adjuvants, B. cinerea incidences were significantly reduced (incidences of 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. In a histopathology study using epifluorescence microscopy, distinct differences were observed in conidium mortality (20.5% vs. 31.2%), germination (60.4% vs. 51.4%) and germ tube lengths (27.8 μm vs. 19.7 μm) between Hydrosilicote and Solitaire in combination with fenhexamid, even though both adjuvants effected similar quantitative and qualitative spray deposition. The study clearly demonstrated the potential of adjuvants to improve the bio-efficacy of a fungicide directly through improved deposition on grapevine leaf surfaces, although bio-efficacy might be influenced by adjuvant mode of action in some cases.

The genus Phaeoacremonium is associated with opportunistic human infections, as well as stunted growth and die-back of various woody hosts, especially grapevines. In this study, Phaeoacremonium species were isolated from necrotic woody tissue of Prunus spp. (plum, peach, nectarine and apricot) from different stone fruit growing areas in South Africa. Morphological and cultural characteristics as well as DNA sequence data (5.8S rDNA, ITS1, ITS2, β-tubulin, actin and 18S rDNA) were used to identify known, and describe novel species. From the total number of wood samples collected (257), 42 Phaeoacremonium isolates were obtained, from which 14 species were identified. Phaeoacremonium scolyti was most frequently isolated, and present on all Prunus species sampled, followed by Togninia minima (anamorph: Pm. aleophilum) and Pm. australiense. Almost all taxa isolated represent new records on Prunus. Furthermore, Pm. australiense, Pm. iranianum, T. fraxinopennsylvanica and Pm. griseorubrum represent new records for South Africa, while Pm. griseorubrum, hitherto only known from humans, is newly reported from a plant host. Five species are newly described, two of which produce a Togninia sexual state. Togninia africana, T. griseo-olivacea and Pm. pallidum are newly described from Prunus armeniaca, while Pm. prunicolum and Pm. fuscum are described from Prunus salicina.

Coniothyrium-like fungi are common wood and soil inhabitants and hyperparasites on other fungi. They belong to different fungal genera within the Pleosporales. Several isolates were obtained on wood of different Prunus species (plum, peach and nectarine) from South Africa, on Actinidia species from Italy and on Laurus nobilis from Turkey. Morphological and cultural characteristics as well as DNA sequence data (5.8S nrDNA, ITS1, ITS2, partial SSU nrDNA) were used to characterise them. The isolates belonged to three species of the recently established genus Paraconiothyrium. This is the first report of Paraconiothyrium brasiliense on Prunus spp. from South Africa. Two new species are described, namely Paraconiothyrium variabile sp. nov. on Prunus persica and Prunus salicina from South Africa, on Actinidia spp. from Italy and on Laurus nobilis from Turkey, and Paraconiothyrium africanum sp. nov. on Prunus persica from South Africa. Although other known species of Paraconiothyrium commonly produce aseptate conidia, those of P. africanum and P. hawaiiense comb. nov. are predominantly two-celled.

During a survey of Prunus wood from South Africa, isolations were made of three presumably Calosphaerialean fungi that formed hyphomycetous, phialidic anamorphs in culture. In order to reveal the phylogenetic relationship of these fungi, they were characterised on a morphological and molecular (LSU and ITS rDNA) basis. Two isolates that formed a teleomorph in culture are newly described as Calosphaeria africana sp. nov. Although asci of Calosphaeria are characterised by having non-amyloid apical rings, two functional wall layers were observed in asci of C. africana, which has hitherto not been observed in any member of the Calosphaeriaceae. However, Calosphaeriaceae (Calosphaeriales, Sordariomycetes) are not closely related to other bitunicate fungi like Dothideomycetes, Chaetothyriales and bitunicate lichens. Possession of two separating wall layers is considered to be a result of both inherited abilities and convergent evolution under a strong selection pressure of the environmental conditions that favour an extension of the ascus. The other two species represented a separate lineage within Calosphaeriaceae, and formed phialophora-like anamorphs. By obtaining the teleomorph in culture, one of them could be identified as a species of Jattaea, described here as Jattaea prunicola sp. nov., while the second, which only produced the anamorph, is named as Jattaea mookgoponga sp. nov. These findings suggest that some species of Jattaea are true members of the Calosphaeriaceae, though the phylogenetic relation of the type, J. algeriensis, remains unknown. Furthermore, it also represents the first report of Jattaea on Prunus wood, and from South Africa.

SUMMARY Plasmopara viticola populations in South Africa were studied for two consecutive grape growing seasons, in an organically managed and a conventional fungicide-sprayed vineyard. Three to four samplings in each season were genotyped with four microsatellite markers (GOB, CES, ISA and BER). Population differentiation (Fst) between the conventional fungicide-sprayed vineyard and organically managed vineyard was low (0.004 and 0.016) in both growing seasons, suggesting one metapopulation. However, differences in the relative contribution of the predominant and new genotypes to epidemics in the two vineyards suggested that fungicide applications may have selected for reduced pathogen diversity. In both years and vineyards, sexual (oosporic) reproduction and/or migration occurred throughout the year and contributed between 12 and 74% to the epidemic. Hardy–Weinberg analyses suggest that South African P. viticola populations are randomly mating. Epidemics in both years and vineyards were dominated by one or two genotypes that each contributed between 14 and 67% to the epidemic through asexual reproduction. The remaining genotypes showed low levels of asexual reproduction, with most genotypes never being able to reproduce asexually. However, for some genotypes asexual reproduction was important, as it enabled survival of the genotypes from one season to the next. In total, ten genotypes were able to survive asexually or vegetatively from one season to the next. The populations were further characterized by the presence of a high frequency of isolates that most likely have elevated ploidy levels.

Black foot disease of grapevines is a decline and dieback disease caused by a soilborne pathogen complex including Cylindrocarpon liriodendri, C. macrodidymum, Campylocarpon fasciculare and Campyl. pseudofasciculare. These pathogens cause primary infections of roots and basal ends of grafted cuttings in nursery soils. Thirteen fungicides were screened in vitro for mycelial inhibition of these pathogens. Prochloraz manganese chloride, benomyl, flusilazole and imazalil were the most effective fungicides tested, and were subsequently included in semi-commercial field trials. Basal ends of grafted cuttings were dipped in various chemical and biological treatments prior to planting in open-rooted nurseries. Black foot pathogens were not isolated from grafted cuttings prior to planting. Additional treatments involved soil amendments with Tricboderma formulations and hot water treatment of dormant nursery grapevines. Field trials were evaluated after eight months. Isolations from uprooted plants revealed low levels of black foot pathogens in the roots of untreated control plants, and significantly higher levels in basal ends of rootstocks. The incidence of black foot pathogens, as well as that of Petri disease pathogens, was not significantly and/or consistently reduced by the majority of chemical or biological treatments. However, these pathogens were not isolated from uprooted plants that were subjected to hot water treatment. It is therefore recommended that hot water treatment of dormant nursery plants be included in an integrated strategy for the proactive management of these diseases in grapevine nurseries.

Hypotheses to explain diversity among African ungulates focus largely on niche separation along a browser/grazer continuum. However, a number of studies advocate that the browser/grazer distinction insufficiently describes the full extent of dietary variation that occurs within and between taxa. Disparate classification schemes exist because of a lack of uniform and reliable data for many taxa, and failure to incorporate spatio-temporal variations into broader assessments of diet. In this study, we tested predictions for diet and dietary niche separation of African savanna ungulates using stable carbon isotope evidence from faeces for proportions of C3 (browse) to C4 (grass) intake among 19 species from the Kruger National Park, South Africa. Dietary predictions from the literature are confirmed in the case of browsers (black rhinoceros Diceros bicornis, giraffe Giraffa camelopardalis, bushbuck Tragelaphus scriptus, kudu Tragelaphus strepsiceros), mixed-feeders (impala Aepyceros melampus, nyala Tragelaphus angasii), and most grazers (white rhinoceros Ceratotherium simum, Burchell’s zebra Equus burchellii, warthog Phacochoerus africanus, hippopotamus Hippopotamus amphibius, blue wildebeest Connochaetes taurinus, tsessebe Damaliscus lunatus, waterbuck Kobus ellipsiprymnus). In contrast, several species showed results differing from most expectations derived from the available literature, including eland Taurotragus oryx, steenbok Raphicerus campestris, grey duiker Sylvicapra grimmia, buffalo Syncerus caffer, roan antelope Hippotragus equinus and sable antelope Hippotragus niger. Many of these discrepancies can be accounted for by seasonal and/or regional dietary differences. Cluster analysis based on a data matrix that incorporates the extent of spatio-temporal dietary variation among Kruger Park ungulates reveals several distinct categories of feeding preferences that extend beyond a two-edged browser/grazer dichotomy, such as mixed-feeders with a preference for either forage class, and spatial/seasonal shifts between uniform and mixed-feeding styles among variable browsers (e.g. grey duiker) and variable grazers (e.g. buffalo). These results highlight the need for approaches that are sensitive to spatio-temporal variations and the continuity of diet.

Protection of wounds against infection by trunk disease pathogens is the most efficient and cost-effective means to prevent grapevine trunk diseases. Studies done to determine the effectiveness of chemical pruning wound protectants have mostly focused on the control of Eutypa lata. However, other important wound pathogens, such as Phaeomoniella chlamydospora, Phaeoacremonium spp., Phomopsis spp. and species of Botryosphaeriaceae ( including Botryosphaeria and aggregate genera), pose just as significant a threat to sustainable grape production. Fungicide sensitivity studies have been conducted for Pa. chlamydospora, P. viticola and E. lata. However, no such studies have been conducted for the pathogenic species of Botryosphaeriaceae from grapevines in South Africa. Ten fungicides were, therefore, tested in vitro for their efficacy on mycelial inhibition of the four most common or pathogenic species of Botryosphaeriaceae in South Africa, 'B.' obtusa, Neofusicoccum australe, N. parvum and Lasiodiplodia theobromae. Iprodione, pyrimethanil, copper ammonium acetate, kresoxim-methyl and boscalid were ineffective in inhibiting the mycelial growth at the highest concentrations tested ( 20 mu g/mL for copper ammonium acetate, 5 mu g/mL for other agents tested). 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 mu g/mL, respectively. These fungicides, except prochloraz manganese chloride, are registered for use 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 species of Botryosphaeriaceae after fungicide treatment. The bioassay findings were, however, inconclusive due to low and varied re-isolation incidences. Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole can be identified as fungicides to be evaluated as pruning wound protectants in additional bioassays and vineyard trials against species of Botryosphaeriaceae as well as the other grapevine trunk disease pathogens.

Wound protection during all stages of grapevine propagation is of utmost importance to prevent infection of propagation material by decline and dieback pathogens. In semi-commercial nursery trials, grapevine rootstock and scion cuttings were soaked in water (control), chemical or biological sanitation products prior to cold storage, prior to grafting (machine- or hand-grafting) and prior to planting in field nurseries. Natural infection levels in basal ends and graft unions of uprooted nursery grapevines were evaluated 8 months after planting. Total pathogen incidences in the water-treated control plants ranged from 30% in basal ends to 13.5% in graft unions. Phaeomoniella chlamydospora was the most commonly isolated pathogen, followed by Phaeoacremonium, Cylindrocarpon + Campylocarpon, Botryosphaeria and Phomopsis spp. Machine-grafted unions generally had lower pathogen incidences compared with hand-grafted graft unions. In general, repeated soak-treatments of propagation material in the tested products resulted in reduced pathogen incidences in nursery grapevines. However, products containing T. harzianum (Trichoflow-T), hydrogen peroxide (Bio-sterilizer) and 8-hydroxyquinoline sulphate (Chinosol) gave inconsistent results, whereas Bronocide (a blend of halogenated alcohols and water) proved to be a good sterilising agent, but reduced certifiable plant yield significantly. Benomyl (at 100 g/100 l), Sporekill (a patented didecyldimethylammonium chloride formulation at 150 ml/100 l) and captan (at 1000 ml/100 l) were consistently the best treatments as growth parameters were not negatively influenced and pathogen incidences in basal ends and graft unions of uprooted plants were reduced.

Petri disease of grapevine is primarily caused by Phaeomoniella chlamydospora. This pathogen affects mostly young grapevines, but is also implicated in esca disease of older grapevines. Little is known about the disease cycle of this fungus. Infected propagation material was identified as a major means of dissemination of the pathogen. Recently, the pathogen was also detected from soil in South Africa and airborne conidia have been found in vineyards. The aim of this study was to use a molecular detection technique to test different samples collected from nurseries in South Africa at different nursery stages for the presence of P. chlamydospora. A one-tube nested-PCR technique was optimised for detecting P. chlamydospora in DNA extracted from soil, water, callusing medium and grapevine wood. The one-tube nested-PCR was sensitive enough to detect as little as 1 fg of P. 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 360 bp P. chlamydospora specific bands. Subsequent sequence analyses and/or restriction enzyme digestions of all 360 bp PCR bands confirmed that all bands were P. chlamydospora specific, except for five bands obtained from callusing media and one from water. Phaeomoniella chlamydospora was positively detected in 25% of rootstock cane sections collected from mother blocks, 42% of rootstock cuttings and 16% of scion cuttings collected during grafting, 40% of water samples collected after pre-storage hydration, 67% of water samples collected during grafting, 8% of callusing medium samples and 17% of soil samples collected from mother blocks. These media can therefore be considered as possible inoculum sources of the pathogen during the nursery stages.

The genus Pilidiella, including its teleomorphs in Schizoparme, has a cosmopolitan distribution and is associated with disease symptoms on many plants. In the past, conidial pigmentation has been used as a character to separate Pilidiella (hyaline to pale brown conidia) from Coniella (dark brown conidia). In recent years, however, the two genera have been regarded as synonymous, the older name Coniella having priority. To address the generic question, sequences of the internal transcribed spacer region (ITS1, ITS2), 5.8S gene, large subunit (LSU) and elongation factor 1-[alpha] gene (EF 1-[alpha]) were analysed to compare the type species of Pilidiella and Coniella. All three gene regions supported the separation of Coniella from Pilidiella, with the majority of taxa residing in Pilidiella. Pilidiella is characterised by having species with hyaline to pale brown conidia (avg. length[ratio]width >1.5), in contrast to the dark brown conidia of Coniella (avg. length[ratio]width [less-than-or-eq, slant]1.5). Pilidiella diplodiella, which is a pathogen associated with white rot of grapevines, was shown to be an older name for C. petrakii. To delineate species in the P. diplodiella species complex, isolates were also compared based on histone (H3) gene sequences. Analyses derived from these sequence data separated P. diplodiella from a newly described species, P. diplodiopsis. The new species P. eucalyptorum sp. nov. is proposed for isolates formerly treated as C. fragariae and 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 sp. nov. is newly described as anamorph of Schizoparme destruens, which is associated with twig dieback of Eucalyptus spp. in Hawaii. A key based on morphological characteristics is provided to separate the taxa treated in this study

Exploitation and characterization of resistance to the root-knot nematode Meloidogyne incognita (Nematoda, Tylenchida) in soybean (Glycine max L. Merr) Host plant resistance can be used to manage Meloidogyne incognita in soybean. This study identified resistant cultivars and showed a poor correlation between early and late pest and damage assessments. The identification of resistance contributes to soybean breeding programs in South Africa.

The effect of Meloidogyne incognita (Nematoda), on lipoxygenase and peroxidase activity in soybean leaves. The goal of this study was to determine whether or not soybean cultivars, LS5995 and Ibis, have mechanisms in place to protect them from nematode infections. Tests were done for lipoxygenase (LOX) and peroxidase (POD) enzymes. Increased LOX and POD activity indicate defense mechanisms in plants.

The South African citrus industry is strongly focused on exports and South Africa is a signatory member of both the World Trade Organisation Agreement on the application of Sanitary and Phytosanitary Measures and the International Plant Protection Convention. Citrus black spot, caused by Guignardia citricarpa, does not occur in all the South African citrus production areas and, therefore, South Africa has a responsibility to provide those trading partners that have identified G. citricarpa as a regulated pest with reliable information about the distribution of citrus black spot within South Africa. Detection surveys were conducted in citrus production areas in the Western Cape, Northern Cape and Free State Provinces and appropriate diagnostic protocols were used to ensure reliable detection of G. citricarpa. Trees in commercial orchards and home gardens on farms and in towns of 17, 9 and 5 magisterial districts in the Western Cape, Northern Cape and Free State Provinces, respectively, were sampled between 1995 and 2010. Fruit samples were taken during June and July, and leaf samples from November to January. None of the 3060 fruit and leaf samples collected during these surveys tested positive for G. citricarpa. Phyllosticta capitalensis, a non-pathogenic, ubiquitous, endophytic species was, however, detected during these surveys. In compliance with relevant International Standards for Phytosanitary Measures and based on the outcome of these official surveys, these three provinces in South Africa can be recognised as citrus black spot pest free areas.

Hair has been shown to archive a uniquely time averaged signal of endocrine activity, and holds attractive advantages for both laboratory and field research. Prior research has explored the potential of hair hormone analysis to examine hormone–behavior relationships. To date, no research has focused on the potential of the technique to investigate age-related changes or taxon differences in endocrine function. It is known that non-human primate infants of many taxa exhibit high cortisol levels after parturition, which rapidly decline with age. It has also been shown that hypercortisolism generally characterizes platyrrhine (New World monkey) endocrine function. These endocrine trends have been characterized using cortisol levels determined from serum, plasma, and feces. Here we test whether cortisol levels determined from hair recover similar phylogenetic and age related patterns in endocrine function in non-human primates. In order to test whether hair cortisol reflect infant hypercortisolism with significant age-related decline, hair cortisol levels are measured in samples from wild vervet monkeys (Chlorocebus aethiops) and captive Guinea baboons (Papio hamadryas papio), ranging in age from infants through juveniles. Further, in order to test whether platyrrhines exhibit significantly higher hair cortisol levels compared to strepsirrhines and catarrhines, and therefore faithfully recover similar signals as more traditionally used substrates (e.g. serum), hair cortisol levels are quantified in adult female hair samples collected from a broad range of non-human primate taxa. Results confirm that hair cortisol levels accurately reflect known phylogenetic and age related patterns of circulating cortisol levels. Therefore, these results suggest that hair may be an ideal hormone bearing substrate for research focused on the examination of population endocrine profiles, cross-sectional studies of endocrine function and taxon variation in hormone levels, as well as stable behavioral trends.