Whole genome sequencing and phylogenomic analysis show support for the splitting of genus Pythium.

The genus Pythium (nom. cons.) sensu lato (s.l.) is composed of many important species of plant pathogens. Early molecular phylogenetic studies suggested paraphyly of Pythium, which led to a formal proposal by Uzuhashi and colleagues in 2010 to split the genus into Pythium sensu stricto (s.s.), Elongisporangium, Globisporangium, Ovatisporangium (= Phytopythium), and Pilasporangium using morphological characters and phylogenies of the mt cytochrome c oxidase subunit 2 (cox2) and D1-D2 domains of nuc 28S rDNA. Although the split was fairly justified by the delineating morphological characters, there were weaknesses in the molecular analyses, which created reluctance in the scientific community to adopt these new genera for the description of new species. In this study, this issue was addressed using phylogenomics. Whole genomes of 109 strains of Pythium and close relatives were sequenced, assembled, and annotated. These data were combined with 10 genomes sequenced in previous studies. Phylogenomic analyses were performed with 148 single-copy genes represented in at least 90% of the taxa in the data set. The results showed support for the division of Pythium s.l. The status of alternative generic names that have been used for species of Pythium in the past (e.g., Artotrogus, Cystosiphon, Eupythium, Nematosporangium, Rheosporangium, Sphaerosporangium) was investigated. Based on our molecular analyses and review of the Pythium generic concepts, we urge the scientific community to adopt the generic names Pythium, Elongisporangium, Globisporangium, and their concepts as proposed by Uzuhashi and colleagues in 2010 in their work going forward. In order to consolidate the taxonomy of these genera, some of the recently described Pythium spp. are transferred to Elongisporangium and Globisporangium.

Detection of Pseudophaeomoniella globosa, an Olive Trunk Pathogen, on Olive Pruning Debris.

Pseudophaeomoniella globosa has recently been identified as a pathogen contributing to olive trunk diseases in South Africa. Little is known regarding the biology and epidemiology of this pathogen. The aim of this study was to investigate whether olive pruning debris act as an inoculum source of P. globosa in established orchards. A nested species-specific PCR was developed for the detection of this pathogen on 138 samples of pruning debris collected from Paarl (40 wood pieces), Stellenbosch (42 wood pieces), and Worcester (56 pieces) in the Western Cape Province, South Africa. Spore washes were made from the samples (5 to 10 cm in length), after which the nested species-specific primers were used to determine the presence of P. globosa on the wood. P. globosa was detected on 37.5% of the pruning debris collected from Paarl, 61.9% from Stellenbosch, and 39.3% from Worcester. The pruning debris that tested positive for P. globosa were evaluated visually by microscopic observations for P. globosa pycnidia. Dark-brown to black pycnidia were found. Conidia from these pycnidia were measured, cultured, and confirmed as P. globosa by sequencing the internal transcribed spacer region. In this study, the pruning debris in established olive orchards were identified as inoculum sources of P. globosa. This study emphasizes the importance of additional means focused on reducing the inoculum sources of this pathogen in these orchards as an additional management strategy against olive trunk diseases.

Pathogenicity Testing of Fungal Isolates Associated with Olive Trunk Diseases in South Africa.

A recent olive trunk disease survey performed in the Western Cape Province, South Africa, identified several fungi associated with olive trunk disease symptoms, including species of Basidiomycota, Botryosphaeriaceae, Coniochaetaceae, Calosphaeriaceae, Diaporthaceae, Diatrypaceae, Phaeomoniellaceae, Phaeosphaeriaceae, Symbiotaphrinaceae, Togniniaceae, and Valsaceae. Many of the species recovered had not yet been reported from olive trees; therefore, the aim of this study was to determine their pathogenicity toward this host. Pathogenicity tests were first conducted on detached shoots to select virulent isolates, which were then used in field trials. During field trials, 2-year-old olive branches of 15-year-old trees were inoculated by inserting colonized agar plugs into artificially wounded tissue. Measurements were made of the internal lesions after 8 months. In total, 58 isolates were selected for the field trials. Species that formed lesions significantly larger than the control could be considered as olive trunk pathogens. These included Biscogniauxia rosacearum, Celerioriella umnquma, Coniochaeta velutina, Coniothyrium ferrarisianum, isolates of the Cytospora pruinosa complex, Didymocyrtis banksiae, Diaporthe foeniculina, Eutypa lata, Fomitiporella viticola, Neofusicoccum stellenboschiana, Neofusicoccum vitifusiforme, Neophaeomoniella niveniae, Phaeoacremonium africanum, Phaeoacremonium minimum, Phaeoacremonium oleae, Phaeoacremonium parasiticum, Phaeoacremonium prunicola, Phaeoacremonium scolyti, Phaeoacremonium spadicum, Pleurostoma richardsiae, Pseudophaeomoniella globosa, Punctularia atropurpurascens, Vredendaliella oleae, an undescribed Cytospora sp., Geosmithia sp., two undescribed Neofusicoccum spp., and four Xenocylindrosporium spp. Pseudophaeomoniella globosa can be regarded as one of the main olive trunk pathogens in South Africa because of its high incidence from olive trunk disease symptoms in established orchards and its high virulence in pathogenicity trials.

Survey of Trunk Pathogens in South African Olive Nurseries.

Several fungal trunk pathogens are associated with olive trunk diseases in South Africa. Little is known regarding the inoculum sources of these pathogens in the olive industry, and no specific management strategies are in place. The aim of this study was to investigate the status of olive nurseries in South Africa, with regard to the presence of trunk pathogens in olive plant material, to determine whether nursery material can be considered inoculum sources contributing to long-distance dispersal of these pathogens. Isolations were made from asymptomatic cuttings from mother blocks (stage 1), asymptomatic and symptomatic rooted cuttings (stage 2), and 1- to 2-year-old trees (stage 3) of eight cultivars in two nurseries. Known olive trunk pathogens of Nectriaceae, Diaporthaceae, Botrysphaeriaceae, Togniniaceae, Phaeomoniellaceae, and Pleurostomataceae were recovered. Neofusicoccum australe was detected in a single stage 1 cutting. Stage 3 material showed the highest incidence of fungi from these families, with Pleurostoma richardsiae having the highest incidence in both nurseries (82.2 and 36.7% of the 1- to 2-year-old trees). Phaeoacremonium parasiticum was present in 28.9% of the trees from one nursery (stage 3). The remaining pathogens occurred in ≤13.3% of the material. These results indicate that nursery propagation material from mother blocks harbors low levels of trunk pathogens and that additional infections occur during the nursery process. Management strategies should focus on the prevention and elimination of infections in mother blocks as well as during the propagation process to ensure that pathogen-free material is delivered to producers.

Canker and Wood Rot Pathogens Present in Young Apple Trees and Propagation Material in the Western Cape of South Africa.

Canker and wood rot pathogens cause dieback and, in severe cases, the death of young apple trees. Recently, a higher occurrence of cankers was observed on 1-year-old apple trees in the Western Cape Province of South Africa. This study aimed to assess the phytosanitary status of nursery trees and propagation material as possible inoculum sources for canker pathogens. Thirteen 1-year-old apple orchards showing canker or dieback symptoms were sampled. Certified nursery apple trees were collected from four nurseries as well as scion and rootstock mother plant material. Isolations were made from the discoloration observed in the vascular tissue of the plant parts and from asymptomatic material. Possible canker and wood rot species were identified with PCR and sequence comparisons of the relevant gene regions and phylogenetic analyses. Similar canker and wood rot species were isolated from 1-year-old diseased apple trees, nursery apple trees, and the propagation material. Forty-five fungal species associated with canker or wood rot symptoms were identified. The top five most abundant fungal species found causing disease on commercial 1-year-old trees were also found in high numbers causing latent infection in certified apple nursery trees. These species were Didymosphaeria rubi-ulmifolii sensu lato, Diplodia seriata, Schizophyllum commune, Didymella pomorum, and Coniochaeta fasciculata, with D. rubi-ulmifolii sensu lato being the dominant species in both sampling materials. In all, 65% of certified nursery apple trees, 5% of scion shoots used for budding, and 21% of rooted rootstock cuttings from layer blocks had latent infections of canker and wood rot pathogens. Pathogenicity trials were conducted with isolates of 39 species, inoculated onto 2-year-old branches of 14-year-old Golden Delicious trees. All species caused lesions that were significantly longer than the control. This study confirmed the presence of canker and wood rot pathogens in apple propagation material as well as certified nursery apple trees, which will aid the improvement of management practices in nurseries.

Diaporthe nebulae sp. nov. and First Report of D. cynaroidis, D. novem, and D. serafiniae on Grapevines in South Africa.

Diaporthe species cause Phomopsis cane and leaf spot as well as Phomopsis dieback on grapevines. Symptoms of Phomopsis dieback have increasingly been observed over the past few years. In order to assess the current status of Diaporthe on grapevines in the Western Cape Province of South Africa, isolations were made from dormant grafted nursery vines, dormant rootstock canes, and dying or dead spurs of field vines. Cultures identified as Diaporthe based on morphological features were further identified to species level by sequencing the internal transcribed spacers (ITS) 1 and 2 and 5.8S rRNA and, for a representative subsample of isolates, the partial beta-tubulin (tub2) and translation elongation factor 1-alpha (EF1-α) genes. Phylogenetic analysis of the combined ITS, tub2, and EF1-α data revealed nine Diaporthe species associated with grapevines during this survey. One of these represents a new species, D. nebulae sp. nov., and three other species, namely D. novem, D. cynaroidis, and D. serafiniae, are reported on grapevines in South Africa for the first time. Species-specific primers were designed for PCR identification of D. ampelina, D. ambigua, and D. foeniculina. Pathogenicity studies conducted on detached grapevine shoots indicated D. ampelina, D. novem, and D. nebulae sp. nov. as the most virulent species.

Diversity of Diatrypaceae Species Associated with Dieback of Grapevines in South Africa, with the Description of Eutypa cremea sp. nov.

Recent studies in grape-growing areas including Australia, California, and Spain have revealed an extensive diversity of Diatrypaceae species on grapevines showing dieback symptoms and cankers. However, in South Africa, little is known regarding the diversity of these species in vineyards. The aim of this study was, therefore, to identify and characterize Diatrypaceae species associated with dieback symptoms of grapevine in South Africa. Isolates were collected from dying spurs of grapevines aged 4 to 8 years old, grapevine wood showing wedge-shaped necrosis when cut in cross section as well as from perithecia on dead grapevine wood. The collected isolates were identified based on morphological characters and phylogenetic analyses of the internal transcribed spacer region (ITS) and ß-tubulin gene. Seven Diatrypaceae species were identified on grapevine, namely Cryptovalsa ampelina, C. rabenhorstii, Eutypa consobrina, E. lata, E. cremea sp. nov., Eutypella citricola, and E. microtheca. The dying spurs yielded the highest diversity of species when compared with the wedge-shaped necrosis and/or perithecia. C. ampelina was the dominant species in the dying spurs, followed by E. citricola, whereas E. lata was the dominant species isolated from the wedge-shaped necroses and perithecia. These results confirm E. lata as an important grapevine canker pathogen in South Africa, but the frequent association of C. ampelina with spur dieback suggests that this pathogen plays a more prominent role in dieback than previously assumed. In some cases, more than one species were isolated from a single symptom, which suggests that interactions may be occurring leading to decline of grapevines. C. rabenhorstii, E. consobrina, E. citricola, E. microtheca, and E. cremea are reported for the first time on grapevine in South Africa.

Molecular phylogeny and taxonomy of Lagenidium-like oomycetes pathogenic to mammals.

Over the past twenty years, infections caused by previously unrecognised oomycete pathogens with morphological and molecular similarities to known Lagenidium species have been observed with increasing frequency, primarily in dogs but also in cats and humans. Three of these pathogens were formally described as Lagenidium giganteum forma caninum, Lagenidium deciduum, and Paralagenidium karlingii in advance of published phylogenetic verification. Due to the complex nature of Lagenidium taxonomy alongside recent reports of mammalian pathogenic species, these taxa needed to be verified with due consideration of the available data for Lagenidium and its allied genera. This study does so through morphologic characterisation of the mammalian pathogenic species, and phylogenetic analyses. The six-gene phylogeny generally supports the most recent comprehensive classification of Lagenidium with a well-supported Lagenidium clade that includes the mammalian pathogens L. giganteum f. caninum and L. deciduum, and well-supported clades for which the names Myzocytiopsis and Salilagenidium can be applied. The genus Paralagenidium is phylogenetically unrelated to any of the main clades within the class Peronosporomycetes. Close relationships between pathogens of mammals and those of insects or nematodes were revealed. Further characterisation of Lagenidium-like taxa is needed to establish the risk of mammalian infection by pathogens of insects and nematodes.

Two clonal lineages of Phytophthora citrophthora from citrus in South Africa represent a single phylogenetic species.

Phytophthora citrophthora from citrus in eastern Corsica and Spain consists of distinct clonal lineages. In South Africa the extent of genetic variation among citrus-associated P. citrophthora isolates is unknown. This was investigated with isolates from South Africa (n =60), Spain (n =10) and six isolates representing three P. citrophthora groups CTR1, CTR2 and CTR3 previously identified with isozyme polymorphisms (Mchau and Coffey 1994). South African and Spanish isolates belonged to two lineages (G1, G2) based on an internal transcribed spacer (ITS) phylogeny, random amplified microsatellites (RAMS) and random amplified polymorphic DNA (RAPD) profiling. Although combined RAMS and RAPD data identified 14 genotypes, unweighted pair group method with arithmetic mean (UPGMA) analyses grouped the isolates into two clusters corresponding to lineages G1 and G2. Lineage G1 predominated among isolates from South Africa (92%) and Spain (100%). Phylogenetic analyses of the ß-tubulin, cytochrome c oxidase subunit I (COX1) and ITS regions did not support the hypothesis that the two lineages represent distinct phylogenetic species but suggested that isozyme group CTR2 and possibly CTR3 are species distinct from P. citrophthora sensu stricto. Mating-type analyses, using tester strains from groups CTR2 and CTR3 revealed that most G1 lineage isolates (n =57) were sterile but that some were of the A1 mating type (n =8) whereas all G2 lineage isolates were A2 (n =5). The mating-type designation was confirmed with P. capsici tester strains. However, when A1 (G1 lineage) and A2 (G2 lineage including CTR1 reference isolates) mating-type isolates were paired in all possible combinations, no oogonia or antheridia were produced. This suggests that only tester strains P. capsici, CTR2 and CTR3 were able to produce sexual structures and that lineages G1 and G2 are sterile and reproductively isolated, which is supported by molecular data.

Pythium spp. Associated with Rooibos Seedlings, and Their Pathogenicity Toward Rooibos, Lupin, and Oat.

Rooibos (Aspalathus linearis) is an important indigenous crop in South Africa. Oomycetes are a common problem in rooibos nurseries, causing serious losses, but limited information is available on the species involved. Molecular and morphological analyses of 117 oomycete isolates from 19 rooibos nurseries and 33 isolates from 11 native rooibos sites revealed the presence of several Pythium spp., including Pythium acanthicum, P. irregulare, P. mamillatum, P. myriotylum, P. pyrilobum, P. cederbergense, and Pythium RB II, and Phytophthora cinnamomi (native site). Most of the species were identified in nurseries and native rooibos, with Pythium irregulare being the most common species occurring in all nurseries and 46% of the native sites. Phylogenetic analyses of the internal transcribed spacer region of the P. irregulare isolates showed that isolates within this species complex fit into three subclades, of which only two have previously been reported. On rooibos, all species except P. acanthicum and the previously characterized P. cederbergense and Pythium RB II were pathogenic and highly virulent. On lupin and oat, rotation crops in nurseries, the three aforementioned species were also nonpathogenic. All the other oomycete species were pathogenic on lupin but less so than on rooibos. On oat, only P. irregulare, P. myriotylum, and P. pyrilobum were pathogenic. This is the first report of P. mamillatum, P. pyrilobum, and P. myriotylum as pathogens of lupin, and P. irregulare and P. pyrilobum as pathogens of oat. The three nonpathogenic Pythium spp. were able to significantly reduce disease caused by pathogenic species in the less susceptible lupin and oat but not on rooibos. On lupin, the nonpathogenic species enhanced the virulence of Phytophthora cinnamomi.

Pythium cederbergense sp. nov. and related taxa from Pythium clade G associated with the South African indigenous plant Aspalathus linearis (rooibos).

The genus Pythium consists of more than 120 species and is subdivided into 11 phylogenetic clades (A-K) based on internal transcribed spacer (ITS) region sequence data. Pythium clade G contains only seven known species, with most not being well described. Our study characterized 12 Pythium isolates from Aspalathus linearis (rooibos) that fit into clade G. Phylogenetic analyses of the ITS region and a combined phylogeny of four gene regions (ITS, ß-tubulin, COX1 and COX2 [cytochrome c oxidase subunits I, II]) identified five clade G subclades. The rooibos isolates formed two groups, Pythium Rooibos I (RB I) and II (RB II), that clustered into two separate clades within subclade 1. The nine Pythium RB I isolates formed a distinct clade from P. iwayamai and is described here as a new species, Pythium cederbergense sp. nov. The three Pythium RB II isolates had P. canariense and P. violae as their closest relatives and were genetically diverse, suggesting the presence of several new species or a species complex that cannot be resolved with the current data, thus precluding a species description of this group. Morphological analyses showed that P. cederbergense and Pythium RB II were indistinguishable from each other but distinct from known clade G species. Clade G studies are being hampered by imprecise morphological descriptions of P. violae, P. canariense and P. iwayamai and each species being represented by only one isolate. The P. cederbergense and Pythium RB II isolates all were nonpathogenic toward rooibos, lupin and oats seedlings. One oligonucleotide was developed for each of P. cederbergense and Pythium RB II, which was able to differentiate the isolates with DNA macro-array analyses.

Molecular Detection and Quantification of Pythium Species: Evolving Taxonomy, New Tools, and Challenges.

The genus Pythium is one of the most important groups of soilborne plant pathogens, present in almost every agricultural soil and attacking the roots of thousands of hosts, reducing crop yield and quality. Most species are generalists, necrotrophic pathogens that infect young juvenile tissue. In fact, Cook and Veseth have called Pythium the "common cold" of wheat, because of its chronic nature and ubiquitous distribution. Where Pythium spp. are the cause of seedling damping-off or emergence reduction, the causal agent can easily be identified based on symptoms and culturing. In more mature plants, however, infection by Pythium spp. is more difficult to diagnose, because of the nonspecific symptoms that could have abiotic causes such as nutrient deficiencies or be due to other root rotting pathogens. Molecular methods that can accurately identify and quantify this important group are needed for disease diagnosis and management recommendations and to better understand the epidemiology and ecology of this important group. The purpose of this article is to outline the current state-of-the-art in the detection and quantification of this important genus. In addition, we will introduce the reader to new changes in the taxonomy of this group.

Molecular analyses of Pythium irregulare isolates from grapevines in South Africa suggest a single variable species.

The Pythium irregulare species complex is the most common and widespread Pythium spp. associated with grapevines in South Africa. This species complex has been subdivided into several morphological and phylogenetic species that are all highly similar at the sequence level [internal transcribed spacer (ITS) and cytochrome c oxidase (cox) regions]. The complex includes Pythium regulare and Pythium cylindrosporum, which are morphologically distinct, and P. irregulare sensu stricto (s.s.) and Pythium cryptoirregulare, which are morphologically similar. The aim of the current study was to determine whether 50 South African grapevine P. irregulare isolates represented more than one phylogenetically distinct species. The isolates were characterised using nuclear (ITS and ß-tubulin) and mitochondrial (cox1 and cox2) gene region phylogenies and two isozyme loci [glucose-6-phosphate isomerase (Gpi) and malate dehydrogenase (Mdh-1)]. Some of the gene sequence data were difficult to interpret phylogenetically, since some isolates contained two or more polymorphic ITS copies within the same isolate (intra-isolate variation) that clustered into different ITS sub-clades, i.e. the P. irregulare s.s. and P. cryptoirregulare sub-clades. The molecular data furthermore only revealed the presence of one phylogenetic species, P. irregulare. Morphological analyses of a subset of the isolates confirmed that the isolates were P. irregulare, and further showed that the P. cylindrosporum ex-type strain formed typical P. irregulare oogonia, and not the previously reported distinct elongated oogonia. Some of the molecular analyses suggested the occurrence of outcrossing events and possibly the formation of aneuploids or polyploids since (i) the nuclear and mitochondrial gene data sets were incongruent, (ii) polymorphic ITS copies were present within the same isolate, (iii) heterozygosities were observed in the ß-tubulin gene and Gpi and Mdh-1 loci in some isolates and (iv) more than two ß-tubulin alleles were detected in some isolates. Altogether, the data suggest that P. irregulare, P. cryptoirregulare, P. cylindrosporum, and possibly P. regulare should be synonimised under the name P. irregulare.

Oogonial biometry and phylogenetic analyses of the Pythium vexans species group from woody agricultural hosts in South Africa reveal distinct groups within this taxon.

Pythium vexans fits into the internal transcribed spacer (ITS) clade K sensu Lévesque & De Cock (2004). Within clade K, P. vexans forms a distinct clade containing two enigmatic species, Pythium indigoferae and Pythium cucurbitacearum of which no ex-type strains are available. In South Africa, as well as in other regions of the world, P. vexans isolates are known to be heterogeneous in their ITS sequences and may consist of more than one species. This study aimed to investigate the diversity of South African P. vexans isolates, mainly from grapevines, but also citrus and apple using (i) phylogenetic analyses of the ITS, cytochrome c oxidase (cox) I, cox II, and ß-tubulin regions and (ii) seven biometric oogonial parameters. Each of the phylogenies clustered P. vexans isolates into a single well-supported clade, distinct from other clade K species. The ß-tubulin region was phylogenetically uninformative regarding the P. vexans group. The ITS phylogeny and combined cox I and II phylogenies, although each revealing several P. vexans subclades, were incongruent. One of the most striking incongruences was the presence of one cox subclade that contained two distinct ITS subclades (Ib and IV). Three groups (A-C) were subjectively identified among South African P. vexans isolates using (i) phylogenetic clades (ITS and cox), (ii) univariate analysis of oogonial diameters, and (iii) multivariate analyses of biometric oogonial parameters. Group A is considered to be P. vexans s. str. since it contained the P. vexans CBS reference strain from Van der Plaats-Niterink (1981). This group had significantly smaller oogonial diameters than group B and C isolates. Group B contained the isolates from ITS subclades Ib and IV, which formed a single cox subclade. The ITS subclade IV isolates were all sexually sterile or produced mainly abortive oospores, as opposed to the sexually fertile subclade Ib isolates, and may thus represent a distinct assemblage within group B. Although ITS subclade Ib included the P. indigoferae ex-type sequence, this group was considered to be P. vexans since South African isolates in this clade produced globose sporangia. Group C contained four apple isolates that were related to, but distinct from P. cucurbitacearum. Although P. vexans groups A-C might be distinct species, they are not described here as such due to (i) these groups only representing some of the known diversity in P. vexans, (ii) conflicting gene tree phylogenies preventing phylogenetic species identification, and (iii) sexually sterile isolates preventing the broad application of biometrical data.