Verticillium dahliae is an economically important soilborne pathogen of tomato of which the microsclerotial inoculum can survive in the soil for extended periods of time. Previous studies, including two studies on tomato, reported that pre-plant microsclerotia soil densities can sometimes, but not always, predict the incidence or severity of Verticillium wilt. The overall objective of the study was to determine if V. dahliae pre-plant microsclerotia densities can be used as a predictive tool for the development of Verticillium wilt of tomatoes. A published qPCR assay was optimised and could detect 4.20 fg V. dahliae DNA (0.16 microsclerotia/g soil) from tomato field soils. There were no noticeable variations in the average number of microsclerotia per hectare across five commercial tomato fields, regardless of whether a high density (four composite samples from 20 soil cores/ha), medium density (four composite samples from 12 soil cores/ha), or low density (one composite sample from five soil cores/ha) sampling method was employed. A highly intensive sampling strategy (20 independently analysed soil cores/ha) was investigated for its predictive value in disease development since it allowed for correlation analyses to be conducted. A significant correlation (r = 0.578, P = 0.008) was evident between the number of microsclerotia pre-plant and disease severity during the season in only one of the three investigated fields; no correlation existed with yield. A glasshouse plant bioassay, using specific quantities of inoculated microsclerotia, established that the microsclerotia threshold required for significant disease development was five microsclerotia/g soil (283.8 fg DNA/g soil). However, one and two microsclerotia/g soil, although not causing significant symptoms, were able to sometimes infect the plants. This study showed that pre-plant V. dahliae soil densities do not have a disease predictive value in tomato, and that plant infection can develop at very low soil inoculum densities.

Phytophthora cinnamomi is a destructive soilborne pathogen causing Phytophthora root rot on avocados worldwide. Little is known about the effect of root sampling time, root quantification method (quantitative real-time PCR [qPCR] versus baiting), and tree sample pooling strategies on the quantification of the pathogen in roots in avocado orchard trees. This was investigated in six avocado orchards in two climatically different production regions (Mooketsi and Letaba) in the Limpopo Province, South Africa, over a 2-year period. Two different tree sample pooling strategies, consisting of either a four-pooled group (four groups each containing five pooled trees) or a single-pooled group (20 trees pooled) per 1 ha, were both shown to be suitable for quantifying P. cinnamomi in tree roots using qPCR or root baiting. P. cinnamomi root quantities from the two tree sample pooling strategies were significantly correlated for both quantification methods. Both quantification methods were suitable for quantifying the pathogen in roots, although qPCR was superior to root baiting at identifying significant differences in P. cinnamomi quantities among root sampling time points. The effect of sampling time was dependent on the investigated year. In 2017, root quantities, which were only evaluated using qPCR, did not reveal a consistent trend of a specific sampling time yielding the highest root quantities for most of the orchards. However, five of the orchards in 2018, based on the qPCR analyses, contained significantly higher P. cinnamomi root quantities in May (late autumn) than in March (early autumn), August (late winter), and October/November (late spring). In 2018, P. cinnamomi root DNA quantities were significantly positively correlated with the number of soil temperature hours at 20 to 24 and 20 to 29°C 2 months preceding the root sampling dates and negatively correlated with the number of hours at 15 to 19°C 2 months preceding root sampling. Our study has identified P. cinnamomi root quantification methods and tree sample pooling strategies, which will be useful for understanding the biology of the pathogen and when disease management strategies should be in place.

In South Africa, potato (Solanum tuberosum) late blight epidemics from 1996 to 2007 were caused by Phytophthora infestans clonal lineage US-1 (McLeod et al. 2001; Pule et al. 2013). Similarly, surveys on tomatoes in the mid-1990s only identified the US-1 clonal lineage in South Africa (McLeod et al., 2001). On potatoes, populations from the Southern Cape and Western Cape regions consisted of persistent mefenoxam-resistant populations (McLeod et al. 2001; Pule et al. 2013). Limited mefenoxam (R-enantiomer of metalaxyl) screening in 2021 in the Western Cape showed that potato isolates were sensitive, which prompted our study. Potato late blight samples were collected in 13 potato fields in the 2021 to 2023 seasons in the Western Cape (n = 4), Free State (n = 7), Limpopo (n = 1) and Kwazulu-Natal (n = 1) Provinces, and one tomato sample in 2022 in the Limpopo Province. Fourteen samples, one per field, were simple sequence repeat (SSR) genotyped for 12 loci (Li et al. 2013) using as DNA template, FTA cards, or genomic DNA extracted from cultures. P. infestans isolations from lesions and DNA culture extractions were conducted as previously described (Pule et al. 2013). SSR genotyping revealed that all 14 P. infestans samples belonged to clonal lineage EU_23_A1 (EU23), which has a phenotype (A1 and metalaxyl sensitive) and SSR genotype matching the US-23 lineage (Saville et al., 2021). As expected, minor polymorphisms were detected among the samples at loci Pi02, G11, D13 and SSR4. Mefenoxam sensitivity testing of seven potato isolates from the Free State (n = 3) and Western Cape (n = 4), and one tomato isolate was conducted as previously described (Mcleod et al. 2001). All isolates were sensitive to mefenoxam since no infection and sporulation occurred at 3 µg/ml. This was expected since EU23 has been reported as mefenoxam sensitive in other countries (Kawchuk et al., 2011; McGrath et al., 2015). Replacement of the US-1 clonal lineage by EU23 suggests that the latter lineage is more aggressive or fit than US-1, but this must be verified especially on potatoes. On tomatoes, on the other hand, EU23 is known as a highly aggressive lineage (Kawchuk et al., 2011; McGrath et al., 2015; Saville et al., 2021). Therefore, population displacements may have first occurred on tomatoes from where the lineage spread to potatoes. In the Cape coastal potato production regions, population displacement may have been supported by the withdrawal of mefenoxam/metalaxyl from the region since 1996 because the EU23 lineage is mefenoxam sensitive, as opposed to the previously prevailing US-1 mefenoxam-resistant lineage. More severe potato late blight epidemics has not been observed in recent years in South Africa. However, tomato late blight has increased and is more prevalent in the Limpopo province. The source of the introduction of EU23 into South Africa is unknown. Only test-tube plants and/or greenhouse tubers may be imported into South Africa since 1997. Therefore, the illegal importation of planting material may have introduced the new genotype. Whether this could have occurred from neighbouring African countries is unknown since P. infestans genotyping has not been conducted in these countries. In Africa, EU23 has been reported in northern African countries (Tunisia, Algeria and Egypt) (Saville et al., 2021; El-Ganainy et al., 2023). Mefenoxam and metalaxyl applications will likely be effective again in the Western Cape, but more samples will have to be tested to confirm this. This will provide growers with a more cost-effective fungicide (metalaxyl) since alternative actives with comparable systemic and curative activity are more expensive.

Phytophthora cinnamomi is an important soilborne pathogen causing root rot and stem cankers on macadamia in South Africa and other countries. It is unknown whether a temporal pattern exists in the colonization of macadamia tree roots by P. cinnamomi. To investigate this, three macadamia orchards were studied bimonthly for 2 years. The study also examined whether two types of feeder roots contained varying levels of P. cinnamomi DNA. A new sensitive P. cinnamomi-specific quantitative real-time PCR (qPCR) assay was used to conduct the assessments. Only symptomatic trees were studied in two of the orchards (ZC and ZD), while symptomatic and asymptomatic trees were assessed in the third orchard (ZA). In the ZC and ZD orchards, white non-suberized feeder roots from a new root flush had higher levels of P. cinnamomi DNA than suberized feeder roots. In orchard ZD, autumn and winter yielded the highest P. cinnamomi root quantities in suberized roots. The symptomatic and asymptomatic trees in orchard ZA generally contained higher P. cinnamomi root quantities in winter than in summer. However, in orchard ZC, no significant temporal pattern was evident, although a trend was noticeable towards higher P. cinnamomi root colonization in autumn, winter and spring. Principal component analyses supported autumn and winter as the months yielding the highest P. cinnamomi root colonization levels. The study has contributed to a better understanding of the temporal nature of P. cinnamomi root colonization, and feeder root types that are optimal for qPCR quantifying the pathogen in macadamia.

Citrus slow decline is a slow developing disease occurring in citrus orchards rendering the citrus orchards uneconomical in a decade or two. Causal agents have not been identified and therefore a study was initiated to identify parameters or combination of parameters that could be useful in early detection of the disease. Four citrus orchards in Mpumalanga Province, South Africa, were identified where citrus slow disease decline was present. Twenty trees of each of the three disease categories including healthy, slight sick and sick trees, were randomly selected and sampled in each orchard. Among the many parameters measured, nematode communities were evaluated as they might show differences between the three tree categories. Results showed that Tylenchulus semipenetrans was the most abundant nematode species present in the three tree categories making up more than 90% of the nematode communities. Nine other plant-parasitic and 21 free-living nematodes were identified among the three tree categories with Acrobeloides being the most abundant nematode genus followed by Prismatolaimus . Among the feeding groups, 73% belonged to bacterivores followed by about 10% to the fungivores. Metabolic foot prints for the three tree categories were very small and closely together in the depleted and degraded quadrat.

Phytophthora root rot (PRR) caused by Phytophthora cinnamomi, is a serious disease and an important constraint to avocado production. The current method of PRR detection in avocado trees is based on a visual assessment of canopy health performed by trained expert personnel with substantial experience. This method is time-consuming if large areas need to be monitored and can be inaccurate in practice due to gradual visual change and spatial variation in the orchards. Additionally, it is important that the infection ratings are consistent and comparable across orchards and growing seasons thereby increasing the complexity of the method. This study aimed to identify the spectral bands that are specific to PRR declining trees when hyperspectral imaging technology is used. Hyperspectral imaging combines digital imaging with spectroscopy, recording contiguous spectral information for each pixel in the image. In the field experiment, avocado trees with varying levels of PRR severity were identified (low, medium, and high levels of infection) whereby the leaves were collected and analysed using hyperspectral imaging in the visible near-infrared - VNIR (400-1000 nm) and short-wave infrared - SWIR (950-2500 nm) spectral range. The identification of relevant bands using hyperspectral image analysis is an important first step for implementing the PRR scouting at block scale an even larger scale, entire orchard. This information can be complemented with remote sensing platforms (satellites or unmanned aerial vehicles) and associated computer vision techniques to present a beneficial alternative to the human vision for the management of PRR infections. Additionally, this will allow for cost-effective and time-saving processes for monitoring large areas of cultivated land enabling the grower to make better PRR infection management decisions.

In the Limpopo Province of South Africa, the sustainable production of tomatoes is threatened by a soilborne disease known as Verticillium wilt. Limited information is available regarding the Verticillium species involved, their genetic diversity and aggressiveness. Therefore, a pathogen survey was conducted in the three major tomato production regions in Limpopo (six fields in the Lowveld and two fields each in the Highveld and Soutpansberg). Verticillium dahliae was identified as the only Verticillium species associated with Verticillium wilt of tomatoes. Conventional vegetative compatibility group (VCG) testing identified 38 isolates as VCG 4B and two isolates as VCG 2B. Pathotype-genotype specific PCR analysis indicated that all the isolates belonged to the non-defoliating pathotype, specifically genotype C. These characteristics allowed for the classification of almost all the isolates from the Lowveld, Highveld and Soutpansberg regions into clonal lineage 4B (38 isolates). The exception was for two isolates from the Lowveld that belonged to lineage 2B(824). Based on a race-specific PCR analysis, all 40 isolates belonged to race 2. Sequencing of the race-specific PCR amplicon revealed the presence of two haplotypes namely I and II. Evaluation of seven inoculation methods (agar plug, millet, node-inoculation, root-dip, sand-bran, soil-drench and toothpick methods) using three isolates showed that the root-dip method was the only method that consistently identified all the isolates as pathogenic based on disease severity as well as an increase in plant height. Assessment of the aggressiveness of the 40 characterised V. dahliae isolates showed that the isolates varied in aggressiveness to the cv. Floradade.

Honeybush (Cyclopia spp.) is an indigenous, leguminous member of the Cape fynbos biome growing in the coastal winter rainfall districts of the Western and Eastern Cape Provinces of South Africa (Joubert et al. 2011). Honeybush is used for the production of herbal teas and is harvested from wild-growing and cultivated plantations (du Toit et al. 1998). Very little is known regarding diseases caused by pathogens on this indigenous plant. Only one report of twig dieback on honeybush caused by several Diaporthe Nitschke species have been reported in South Africa (Smit et al. 2021). Several honeybush producers reported poor growth and dieback in their C. subternata plantations in the Western Cape Province, South Africa. Symptoms included twig dieback, branch dieback, death of branches as well as death of entire plants. In April 2008, branches from 8-year-old cultivated plants with dieback symptoms were collected in Stellenbosch. Fungal isolations were carried out from affected material as described by Van Niekerk et al. (2004) which consistently revealed the presence of a Botryosphaeriaceae species. Two isolates were grown on water agar with sterile pine needles and incubated at 25˚C using a 12-hour day/night cycle and near-ultraviolet light. Pycnidia formed after two weeks. Morphological characteristics similar to Neofusicoccum australe (Slippers, Crous & Wingfield) Crous, Slippers & Phillips were observed (Phillips et al. 2013). Conidia were hyaline, aseptate, fusiform with subtruncate bases (16.8-)18.8-22.1(-24.6) × (4.8-)5.3-6.1(-6.4) µm (n=50). Conidiogenous cells were holoblastic, hyaline and subcylindrical to flask-shaped tapering to the apex (11-15 × 2 µm) (n=10). Colonies on potato dextrose agar were light primrose turning olivaceous grey after 7 days with a light-yellow pigment diffusing into the medium. Mycelia was moderately dense with an appressed centre mat. The identity of the isolates was further confirmed by sequencing the ribosomal RNA Internal Transcribed Spacer (ITS) and the elongation factor 1-alpha (EF-1α) gene regions using primer pairs ITS4-ITS5 (White et al. 1990) and EF1-728F-EF1-986R (Alves et al. 2008), respectively. Sequences had a 100% similarity to N. australe ex-type CMW6837 isolate (accessions AY339262 and AY339270) (Slippers et al. 2004). Two isolates (STEU6554 and STEU6557) were deposited in the culture collection at the Department of Plant Pathology at Stellenbosch University and the sequences were submitted to GenBank with accession numbers ON745603, ON745604, ON746573 and ON746574. Pathogenicity tests using the two N. australe isolates were conducted by inoculating two shoots each of three field-grown C. subternata plants with a 4mm colonised potato dextrose agar (PDA) mycelium plug of each isolate on wounds made by a 4mm cork borer (Van Niekerk et al. 2004). A third shoot was inoculated with a uncolonized PDA plug as the negative control. After 12 weeks, brown-black lesions that were significantly longer (average 55.2 mm) than the uncolonized agar plug control (16.1 mm) were observed. Lesions were observed in all three plants. Neofusicoccum australe was re-isolated (van Niekerk et al. 2004) from all inoculated shoots confirming Koch's postulates. The economic impact and damages caused by N. australe as well as its incidence and severity on honeybush in South Africa is unknown. However, the pathogen caused dieback of entire branches and death of plants indicating that it could be an important pathogen of honeybush. Additionally, N. australe is one of the most important disease-causing Botryosphaeriaceae pathogens on a wide range of economical fruit and vine crops globally (Mojeremane et al. 2020). This is the first report of N. australe as a known pathogen causing decline and dieback of C. subternata in South Africa. References: Alves, A. et al. 2008. Fungal Divers. 28:1. du Toit, J. et al. 1998. J. Sustain. Agric. 12:67. Joubert, E. et al. 2011. S. Afr. J. Bot. 77:887. Mojeremane, K. et al. 2020. Phytopathol. Mediterr. 59:581. Phillips, A. J. et al. 2013. Stud. Mycol. 76:51. Slippers, B. et al. 2004. Mycologia 96:1030. Smit, L. et al. 2021. Eur. J. Plant Pathol. 161:565. van Niekerk, J. M. et al. 2004. Mycologia 96:781. White, T. J. et al. 1990. Pages 315 in: In PCR Protocols: A Guide to Methods and Applications. Academic Press Inc, USA. Declaration. The author(s) declare no conflict of interest Acknowledgments. This work benefitted from the financial support of the Agricultural Research Council, Infruitec-Nietvoorbij, South Africa.

Citrus black spot (CBS), caused by Phyllosticta citricarpa, is a disease that affects citrus worldwide. In different regions of the world where both mating types occur, reports differ as to whether asexually produced pycnidiospores play an important role in the epidemiology of CBS and fruit infections. Therefore, we investigated the potential role of pycnidiospores in two lemon orchards in South Africa by using microsatellite-based analysis of fruit populations over time (two seasons) and space (distance). The two orchards were situated in the semiarid North West province (NW) and subtropical Mpumalanga province (MP). Each population contained both mating types in 1:1 ratios, and linkage disequilibrium analysis indicated a random mating population. A total of 109 and 94 multilocus genotypes (MLGs) were detected across the two seasons in the NW and MP orchards, respectively. Psex analyses indicated that most MLGs probably resulted from sexual reproduction, but there were six predominant MLGs in each orchard that were probably replicated via asexual reproduction. Each of the predominant MLGs was monomorphic for mating type. In the NW, five predominant and widespread MLGs caused 46 and 44% of the fruit infections in the two seasons, whereas in MP, three MLGs caused 34 and 48% of the infections. Asexual reproduction in both orchards was supported by low MLG evenness values in all populations. In both orchards, distance was not a reliable predictor of population genetic substructuring or season. Populations of P. citricarpa in the MP and NW orchards were significantly genetically differentiated from each other.

Phytophthora root rot, caused by Phytophthora cactorum, is an economically important disease on young apple trees. Limited information is available on the effect of different phosphonate application methods and dosages on disease control, fruit and root phosphite concentrations, and soil and root pathogen inoculum levels. Evaluation of phosphonate treatments in three apple orchard trials (two in the Grabouw and one in the Koue Bokkeveld region) showed that foliar sprays (ammonium or potassium phosphonate), trunk sprays and trunk paints, were equally effective at increasing trunk diameter in one trial and yield in a second trial over a 25-month period. Foliar ammonium and potassium phosphonate sprays (12 g of phosphorous acid/tree), and two different dosages of the ammonium phosphonate sprays (∼4.8 g or 12 g of phosphorous acid/tree) were all equally effective at improving tree growth. The addition of a bark penetrant (polyether-polymethylsiloxane-copolymer) to trunk sprays did not improve the activity of trunk sprays. The low dosage ammonium phosphonate foliar spray (∼4.8 g a.i./tree) was the only treatment that, in general, yielded significantly lower root phosphite concentrations than the other phosphonate treatments. Root phosphite concentrations were significantly positively correlated (P < 0.0001) with an increase in trunk diameter and negatively (P < 0.0001) with P. cactorum root DNA quantities. Phosphite fruit residues were <31 ppm for all treatments, with the trunk paint treatment (80 g of phosphorous acid/tree applied annually) yielding significantly lower residues than the higher dosage foliar sprays (∼12 g a.i./tree). Twenty-one months posttreatment, most of the phosphonate treatments in all of the trials similarly significantly reduced P. cactorum DNA quantities estimated directly from roots, but not from soil based on soil baiting DNA analysis. Pathogen quantities in fine feeder roots did not differ significantly from those in higher-order roots (<5 mm diameter). P. cactorum DNA quantities estimated using DNA quantification directly from roots were significantly correlated (P < 0.0001) with those obtained through root leaf baiting DNA analysis and, to a lesser extent, with soil leaf baiting DNA quantities (P = 0.025).

Common scab of potato is an economically important soilborne disease caused by various Streptomyces species. The disease is very difficult to control, but biofumigation has recently shown some potential. Biofumigation consists of the incorporation of Brassica spp. crop residues containing glucosinolates that upon cell disruption are hydrolysed by the enzyme myrosinase to yield a diversity of biologically-active hydrolysis products, of which volatile isothiocyanates are the most toxic to soil microbes. In this study common scab was significantly reduced through soil incorporation of fresh and air-dried residues of Brassica oleracea var. capitata (cabbage) when applied prior to two consecutive potato plantings. The in vitro effect of volatile emissions from various Brassica species on pathogenic and non-pathogenic Streptomyces isolates was also evaluated using a bioassay method. In the chamber bioassay freshly macerated Brassica tissue, (B. oleracea var. capitata and B. juncea/S. alba mix) suppressed sporulation but not hyphal growth of the 79 evaluated Streptomyces isolates. The chamber bioassay also showed that the Brassica tissue volatiles were bacteriostatic, since isolates re-grew when removed from the chamber and transferred to fresh media. The results suggest that soil biofumigation could significantly influence composition of the Streptomyces community in agricultural soils and lead to significant suppression of common scab on potato.

Phosphonic acid-based fungicides, also referred to as phosphonates, have been used extensively as crop protectants in horticulture since the late 1970s, and more recently in native ecosystems and forestry. Discovering that phosphonates are effective against foliar and soilborne oomycete diseases, such as those caused by species of Phytophthora, Pythium and Plasmopara, was a significant breakthrough, especially for soilborne pathogens that are notoriously difficult to manage. Phosphonates have played an important role in protection of forests and sensitive natural ecosystems, under threat from these pathogens. Since introduction, their increased application in management of non-oomycete diseases, along with other functionalities, demonstrates their versatility in agriculture and more broadly. Continued use of phosphonic acid crop protectants will be underpinned by demonstrated efficacy and safety, and a better understanding of specific interactions within the plant, pathogen and environment. (c) 2020 Society of Chemical Industry

In South Africa, phosphonate fungicides are widely used in avocado in a preventative management strategy against Phytophthora root rot (PRR), caused by Phytophthora cinnamomi. Trunk injections, curative and preventative (0.3-g a.i./m2 and 0.5-g a.i./m2 of canopy area respectively), done as two annual applications (fall and summer) are widely used to manage PRR. However, these applications have become problematic due to frequent exceedances of the European Union (EU) maximum residue level (MRL) of phosphonic acid (synonym phosphite) in fruit. Our study showed that fruit residues can be reduced by the use of specific phosphonate application methods (foliar versus trunk injections), and application timing (fall versus summer). In the 2016/17 season, trunk injections applied only in fall (1-g a.i./m2 of canopy area), or applied at the registered curative split dosage in fall (0.5-g a.i./m2) and summer (0.5-g a.i./m2), resulted in exceedances or near exceedances of the MRL for some replicates among treatments. In comparison, 3% a.i. ammonium- or potassium phosphonate foliar sprays applied only in fall had fruit residues that were consistently well below the EU MRL. The 2% a.i. foliar sprays, specifically the ammonium phosphonate formulation, done as a split application in fall (1.5% a.i) and summer (0.5% a.i.) sometimes resulted in significantly higher fruit residues than fall only applications. Some replicates of this treatment nearly exceeded the MRL. In the 2017/18 season, 2% and 2.5% a.i. foliar sprays (ammonium- and potassium phosphonate) applied only in fall yielded fruit residues that were consistently below the MRL. In contrast, the registered split dosage trunk injections (curative and preventative) sometimes resulted in exceedances of the MRL or near exceedances. In both seasons, foliar sprays at the start of the season yielded phosphite concentrations in roots that did not differ significantly from trunk injections, but towards the end of the season, concentrations were sometimes significantly lower. Foliar sprays applied only in fall have the potential to reduce fruit residues.

Apple replant disease (ARD) is a biological phenomenon that is encountered when old apple orchards are replanted, resulting in tree growth and yield reductions in young trees. Three ARD orchard trials were conducted, which showed that semiselective chemicals (fenamiphos, metalaxyl, imidacloprid, and phosphonates) used independently, two fumigant formulations (33.3% chloropicrin and 60.8% 1,3-dichloropropene [Pic33-1,3D] and 57.% chloropicrin and 38% 1,3 dichloropropene [Pic57-1,3D]), and semiselective chemicals combined with Pic33-1,3D or Pic57-1,3D all contributed to significant increases in tree growth (trunk diameter and shoot length) relative to the untreated control 3 to 4 years postplanting. The treatments did not differ significantly from each other in improving tree growth. Yield was more indicative of treatment efficacy, but this varied between the three orchards. The Pic33-1,3D fumigant in combination with semiselective chemistries was the most consistent in significantly increasing cumulative yields. The Pic57-1,3D treatment was superior in increasing yields relative to the Pic33-1,3D treatment, because (i) it significantly increased cumulative yields in comparison with the Pic33-1,3D treatment in one orchard and (ii) in another orchard, a significant increase in yield was obtained with Pic57-1,3D relative to the control treatment but not with the Pic33-1,3D treatment. The quantification of ARD causative agents 20 months postplant showed that Phytophthora cactorum contributed to disease development in all three orchards; significant negative correlations existed between the quantity of P. cactorum DNA detected in tree roots and tree growth and less often, yield. In two orchards, only some of the treatments that significantly reduced the quantity of P. cactorum DNA in tree roots relative to the control also resulted in a significant increase in tree growth. Some of the aforementioned trends were also evident for Pratylenchus spp. root densities in two of the orchards. There was a significant positive correlation between P. cactorum root DNA quantities and Pratylenchus spp. root densities. Pythium spp. and "Cylindrocarpon"-like DNA quantities detected in tree roots typically were not indicative of treatment efficacy. However, a significant positive correlation existed between these two pathogen groups, suggesting complex interactions not associated with pathogen quantities per se.

Apple scab, caused by Venturia inaequalis, is the most common fruit and foliar disease in commercial apple production worldwide. Early in the production season, preventative contact fungicide sprays are essential for protecting highly susceptible continuously unfolding and expanding young leaves. In South Africa, mancozeb is a key contact fungicide used for controlling apple scab early in the season. The current study developed deposition benchmarks indicative of the biological efficacy of mancozeb against apple scab, using a laboratory-based apple seedling model system. The model system employed a yellow fluorescent pigment that is known to be an effective tracer of mancozeb deposition. A concentration range of mancozeb (0.15 to 1 times the registered dosage) and fluorescent pigment concentrations was sprayed onto seedling leaves, which yielded various fluorescent particle coverage (FPC%) levels. Modeling of the FPC% values versus percent disease control yielded different benchmark values when disease quantification was conducted using two different methods. Thermal infrared imaging (TIRI) disease quantification resulted in a benchmark model where 0.40%, 0.79%, and 1.35 FPC% yielded 50, 75, and 90% apple scab control, respectively. These FPC% values were higher than the benchmarks (0.10, 0.20, and 0.34 FPC%, respectively) obtained with quantitative real-time PCR (qPCR) disease quantification. The qPCR benchmark model is recommended as a guideline for evaluating the efficacy of mancozeb sprays on leaves in apple orchards since the TIRI benchmark model underestimated disease control. The TIRI benchmark model yielded 68% disease control at the lowest mancozeb dosage, yet no visible lesion developed at this dosage. Both benchmark models showed that mancozeb yielded high levels of disease control at very low concentrations; for the qPCR benchmark model the FPC% value of the FPC90 (90% control) corresponded to 0.15 times that of the registered mancozeb concentration in South Africa, i.e., 85% lower than the registered dosage.

Investigations into inoculum sources and disease management strategies require effective pathogen quantification techniques, which should ideally also be reflective of the extent of plant damage. The current study investigated whether determination of relative pathogen DNA quantity in root tissue can improve the assessment of plant damage by several oomycete apple replant pathogens when compared to absolute DNA quantifications and percent roots infected. Published real-time quantitative PCR (qPCR) assays were utilized to quantify pathogen DNA, except for Phytopythium vexans for which a new qPCR assay was developed. Relative pathogen DNA quantifications employed a mutated Escherichia coli gene spiked into the DNA extraction buffer. Pathogen quantifications were not improved through relative DNA quantifications since relative DNA quantities were highly and significantly correlated with absolute pathogen DNA quantities. This was evident from: (i) glasshouse experiments where five oomycete apple replant disease pathogens ( Pythium sylvaticum , Pythium irregulare , Pythium ultimum , P. vexans and Phytophthora cactorum ) were quantified from artificially inoculated apple seedlings roots, and (ii) quantification of P. irregulare from naturally-infected nursery tree roots. Relative- and absolute pathogen DNA quantities in infected glasshouse seedling roots (all five species) and nursery tree roots ( P. irregulare ), were furthermore significantly correlated with percent roots infected. Pathogen root DNA quantities (relative and absolute) obtained from the fine feeder root systems of seedlings from the glasshouse trials were significantly negatively correlated with increase in seedling length for P. sylvaticum, P. vexans and P. ultimum infected seedlings . This, however, was not true for P. cactorum and P. irregulare . The percent infected roots also had a significant negative correlation with increase in seedling length for P. sylvaticum, P. vexans and P. ultimum and P. irregulare , but not for P. cactorum.

Phytophthora cinnamomi Rands is a devastating root rot pathogen of avocado. Robust and sensitive root quantification methods are required for determining seasonal P. cinnamomi root colonization patterns and evaluating management strategies. Our study investigated four P. cinnamomi root quantification methods using a newly developed P. cinnamomi-avocado-seedling bioassay system and a P. cinnamomi-specific probe-based qPCR assay. Phytophthora cinnamomi quantification through plating of roots (root plating) or lemon leaf disks obtained from root baitings (root-baiting-plating) onto semi-selective media were the best methods. Root plating consistently yielded significant differences in P. cinnamomi quantities obtained from seedling roots inoculated with five zoospore concentrations (10-1 × 105 zoospores/ml), whereas root-baiting-plating did so less often. The two methods were comparable in yielding root quantities that were significantly correlated with the inoculated zoospore concentrations, rarely yielding false negatives and having the lowest variability between replicates of the same treatment. qPCR quantification from roots was also an effective method; however, treatment replicates were highly variable and false negatives occurred more frequently. The least effective quantification method was qPCR quantification from lemon leaf disks obtained from root baitings.

Apple replant disease (ARD) is a biological phenomenon caused by soilborne agents that include selected species of fungi ( Rhizoctonia and ‘ Cylindrocarpon’ -like), oomycetes ( Pythium, Phytopythium and Phytophthora ) and nematodes ( Pratylenchus ). Old orchard soils previously planted to apple or related species are a primary inoculum source of ARD pathogens. In the current study, nursery trees and irrigation water were investigated as potential external ARD inoculum sources in South Africa. Investigations conducted at five nurseries over two years revealed that roots of nursery trees were infested by several ARD agents. In the 2013 sampling year, Pythium irregulare and Pythium ultimum were obtained from trees in five and two of the nurseries, respectively, using isolation studies. Based on isolation studies conducted on individual trees in 2013, 47% and 4% of all surveyed trees contained P. irregulare and P. ultimum, respectively. In the 2014 season, real-time quantitative PCR (qPCR) analyses of root tissue from individual trees demonstrated that all of the nurseries and 95% of trees contained P. irregulare , whereas three nurseries and 41% of trees contained P. utlimum . The only other oomycete pathogens that were detected in nursery tree roots were Pythium spp. complex B2A and Pythium sylvaticum and each only occurred in one nursery in one of the sampling years. For all nurseries in both years, trees were consistently infected with ‘ Cylindrocarpon’ -like spp. Pratylenchus spp., which were only analysed in 2013, was present in all five nurseries and in 29% of the trees. Infestation levels were high, with 22% of trees having Pratylenchus root densities that exceeded 100 per 5 g of roots. Pythium irregulare was the dominant oomycete pathogen detected in irrigation water samples (31% to 76% of the samples) obtained from 13 orchards over two years on a monthly basis. The ARD pathogens P. ultimum, Phytopythium litorale and Pythium spp. complex B2A were rarely identified in irrigation water, along with five other non-pathogenic Pythium and Phytopythium species. The association of ARD causative agents with nursery trees and irrigation water suggests that these could function as potential ARD inoculum sources that might limit post-plant tree growth.

Phosphonate fungicides are registered on various tree crops in South Africa for the management of oomycete root rot pathogens, but not on apple trees. The study investigated several phosphonate treatments previously evaluated independently by technical advisors in South Africa. A replicated orchard trial was conducted in non-bearing asymptomatic orchards; tree roots were infected by oomycetes but foliar symptoms were absent. Phosphonate foliar-, trunk paint- and soil drench treatments were equally effective, and consistently resulted in a significant reduction in Phytophthora cactorum and Pythium irregulare root DNA quantities relative to the non-treated control. The latter was not always true for phosphonate trunk spray treatments. Trunk paint applications applied at an annual dosage of 40 g phosphorous acid/tree yielded significantly higher root phosphite (breakdown product of phosphonates) concentrations than the soil drench and trunk spray applications; the latter were applied at lower annual dosages of 7.5 g a.i./tree and 20 g a.i./tree, respectively. Foliar sprays applied at a low annual dosage (1.8 to 3.0 g a.i./tree) often outperformed the soil drench and trunk spray treatments in root phosphite concentrations. No clear association was evident between root phosphite concentrations and pathogen suppression. Root phosphite typically peaked at 8-weeks post-treatment for winter applications, and between 2- to 4-weeks for summer applications. A rapid decline in root phosphite was evident over the 12-week summer period, but not for winter applications. Monitoring root growth in the untreated control plots showed that root growth was continuous but that it peaked in summer, with reduced growth in winter.

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 < 0.001) and high (r = 0.896) between fluorescent particle coverage (FPC%) and mancozeb residue (mg/kgDry Weight) for all five formulations. The particle size ranges of two wettable powder (WP) formulations were significantly smaller than those of the other WP formulations, but this did not result in differences in mancozeb residue on apple leaves. The persistence of mancozeb to different rain volumes applied to apple leaves was determined for three mancozeb formulation treatments: Dithane M-45 800 WP NT, Ventum 800 WP, and Ventum 800 WP combined with the sticker-spreader adjuvant Nu-Film P. There were no significant differences (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%).