Predominant Clonal Reproduction with Infrequent Genetic Recombination of Phaeoacremonium minimum in Western Cape Vineyards.

Phaeoacremonium minimum is an important esca and Petri disease pathogen that causes dieback of grapevines in South Africa. Little is known regarding the reproductive strategy of the pathogen. Sexual reproduction could lead to a better adaptation of the pathogen to disease management strategies by combining alleles through recombination. The study aimed to investigate the genetic diversity and recombination potential of eight populations in the Western Cape, from six commercial vineyards and two nursery rootstock mother blocks. This was achieved by developing and applying nine polymorphic microsatellites and mating-type-specific markers. Thirty-seven genotypes were identified from 295 isolates. Populations were characterised by the same dominant genotype (MLG20 occurring 65.43%), low genotypic diversity (H) and high numbers of clones (81.36% of dataset). However, genotypes from the same sampling sites were not closely related based on a minimum spanning network and had high molecular variation within populations (94%), suggesting that multiple introductions of different genotypes occurred over time. Significant linkage disequilibrium among loci (r̅d) further indicated a dominant asexual cycle, even though perithecia have been observed in these four populations. The two rootstock mother blocks had unique genotypes and genotypes shared with the vineyard populations. Propagation material obtained from infected rootstock mother blocks could lead to the spread of more genotypes to newly established vineyards. Based on our results, it is important to determine the health status of rootstock mother blocks. Management strategies must focus on reducing aerial inoculum to prevent repeated infections and further spread of P. minimum genotypes.

First report of Neofusicoccum australe causing dieback of honeybush in the Western Cape, South Africa.

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.

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.

Characterization and Pathogenicity of Diplodia, Lasiodiplodia, and Neofusicoccum Species Causing Botryosphaeria Canker and Dieback of Apple Trees in Central Chile.

In recent years, the number of apple trees affected by Botryosphaeria cankers and dieback has considerably increased in central Chile. This study aimed to identify the species of Botryosphaeriaceae associated with canker and dieback symptoms, estimate disease incidence and distributions, and study their pathogenicity and virulence on apple and other fruit crops. A field survey of 34 commercial orchards of apple (7 to 30 years old) was conducted in 16 localities, obtaining 270 symptomatic branch and trunk samples in 2017 and 2018 growing seasons. The incidence of Botryosphaeria canker and dieback ranged between 5 and 40%, and a total of 255 isolates of Botryosphaeriaceae spp. were obtained from 238 cankers. Morphological identification along with phylogenetic studies of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of the rDNA, part of the translation elongation factor 1-α (tef1-α), and part of the ß-tubulin (tub2) genes allowed us to identify Diplodia mutila (n = 49 isolates), D. seriata (n = 136 isolates), Lasiodiplodia theobromae (n = 16 isolates), and Neofusicoccum arbuti (n = 54 isolates). L. theobromae was isolated mainly from apple dieback from northern localities. All pathogens tested were pathogenic, causing canker and dieback symptoms on lignified twigs of apple, pear, walnut, and green grapevine shoots in the field. Isolates of N. arbuti were the most virulent, reproducing more severe cankers on the lignified tissues inoculated. This study reports, for the first time, D. mutila and L. theobromae associated with Botryosphaeria canker and dieback in Chile, and it is the first description of N. arbuti causing apple dieback worldwide.

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.

Occurrence of Canker and Wood Rot Pathogens on Stone Fruit Propagation Material and Nursery Trees in the Western Cape of South Africa.

Dieback and canker of young stone fruit trees can cause suboptimal growth and even death under severe conditions. One source of inoculum of canker pathogens could be through nursery trees harboring latent infections that would not be visible to inspections done according to the deciduous fruit scheme. The objectives of this study were to identify the canker and wood rot fungal pathogens present in nursery stone fruit trees as well as in propagation material and to evaluate their pathogenicity. Isolations were made from scion and rootstock propagation material and from certified nursery stone fruit trees. The plant material sampled did not have any external symptoms. The certified nursery trees when cross-sectioned displayed brown discoloration from the pruning wound, the bud union, and often the crown. Fungal species isolated were identified by sequencing of the relevant barcoding genes and phylogenetic analyses thereof. Canker- and wood rot-associated fungi were identified. Buds used for budding had low levels of infection, with 1.2% of dormant buds infected and 0.4% of green buds infected. The dormant rootstock shoots had a canker pathogen incidence of 6.2% before they were planted in the nursery fields and increased inasmuch as the ungrafted, rooted rootstock plants had 11.1% infection with canker and wood rot pathogens. Out of 1,080 nursery trees, the canker- and wood rot-associated fungi infected 21.8% of trees. The canker-causing pathogens that were isolated the most were Cadophora luteo-olivacea and Diplodia seriata. A low incidence of wood rot fungi was found, with only 1.5% of nursery trees infected. In total, 26 new reports of fungal species on stone fruit in South Africa were made. Of these, 22 have not been found on stone fruit worldwide. The pathogenicity trials' results confirmed the pathogenic status of these newly reported species. All of the isolates tested formed lesions significantly longer than the control, 4 months after wound inoculation of 2-year-old shoots of two plum orchards. Lasiodiplodia theobromae was the most virulent species on both plum cultivars. The results of this research showed that nursery stone fruit trees and propagation material can harbor latent infections. Different management practices need to be evaluated to prevent these infections to ensure healthier stone fruit nursery trees.

Investigation of Trichoderma species colonization of nursery grapevines for improved management of black foot disease.

BACKGROUND: Black foot disease (BFD) is one of the main fungal diseases associated with young grapevine decline. Trichoderma holds the potential to be used as biocontrol agent against this disease, though variable success of colonization were found when applied to nursery vines in previous studies. Therefore, field experiments were established to evaluate different methods of application of Trichoderma atroviride, and to evaluate the efficacy of different commercial Trichoderma products on BFD in nursery vines post callusing over two seasons. RESULTS: Only in one season of the trial evaluating different products did all of the Trichoderma treatments significantly lower the black foot infections in the rootstock bases of the vines (mean black foot pathogen incidence of 1.00 to 2.50% in Trichoderma treated vines versus 6.50% in the untreated control). When comparing tissue parts, the base of the vine and collar roots had significantly higher Trichoderma colonization than the middle and root tip parts. Significantly less BFD pathogens were isolated from the base in comparison to the roots. These colonization trends were found for both field trials over both seasons. The different application methods showed that dipping of basal ends in the dry formulation followed by monthly soil drenches consistently gave higher colonization [mean Trichoderma incidence in the bases were 39.20% (2016/2017) and 28.00% (2017/2018)], while the 1 h soak of the bases of vines was not effective [mean Trichoderma incidence in the bases were 8.80% (2016/2017) and 4.00% (2017/2018)] and did not differ from the untreated control. CONCLUSION: Even though Trichoderma spp. were not sufficient to prevent infections by BFD pathogens, a certain degree of protection was obtained in the basal ends, which may contribute to longevity of the vines once planted in the vineyard.

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.

Eutypa, Eutypella, and Cryptovalsa Species (Diatrypaceae) Associated with Prunus Species in South Africa.

Stone fruit trees (Prunus spp.) are economically important fruit trees cultivated in South Africa. These trees are often grown in close proximity to vineyards and are to a large extent affected by the same trunk disease pathogens as grapevines. The aim of the present study was to determine whether stone fruit trees are inhabited by Diatrypaceae species known from grapevines and whether these trees could act as alternative hosts for these fungal species. Isolations were carried out from symptomatic wood of Prunus species (almond, apricot, cherry, nectarine, peach, and plum) in stone fruit growing areas in South Africa. Identification of isolates was based on phylogenetic analyses of the internal transcribed spacer region and ß-tubulin gene. Forty-six Diatrypaceae isolates were obtained from a total of 380 wood samples, from which five species were identified. All five species have also been associated with dieback of grapevine. The highest number of isolates was found on apricot followed by plum. No Diatrypaceae species were isolated from peach and nectarine. Eutypa lata was the dominant species isolated (26 isolates), followed by Cryptovalsa ampelina (7), Eutypa cremea (5), Eutypella citricola (5), and Eutypella microtheca (3). First reports from Prunus spp. are E. cremea, E. citricola, and E. microtheca. Pathogenicity tests conducted on apricot and plum revealed that all these species are pathogenic to these hosts, causing red-brown necrotic lesions like those typical of Eutypa dieback on apricot.

Cylindrocarpon pauciseptatum sp. nov., with notes on Cylindrocarpon species with wide, predominantly 3-septate macroconidia.

A Cylindrocarpon species with up to 10 microm wide, straight and predominantly 3-septate macroconidia, subglobose to ovoidal microconidia and chlamydospores, is described as Cyl. pauciseptatum. It is most similar to Cyl. austrodestructans but no chlamydospores and microconidia are formed in the latter. Similar macroconidia also occur in Cyl. theobromicola, which forms oval to ellipsoidal microconidia at least sparsely and has slightly curved macroconidia, and Cyl. destructans var. crassum, which forms abundant 1-celled microconidia. DNA sequence data of the internal transcribed spacer regions 1 and 2 plus the 5.8S rDNA and the partial beta-tubulin gene were used for phylogenetic inferences. Cylindrocarpon pauciseptatum and Cyl. macrodidymum are monophyletic and are closely related to other species of Cylindrocarpon sensu stricto including members of the Cyl. destructans (teleomorph, Neonectria radicicola) species complex, which accommodates Cyl. liriodendri (teleomorph, Neon. liriodendri), Cyl. destructans var. crassum and Cyl. austrodestructans (teleomorph, Neonectria austroradicicola comb. nov.). Cylindrocarpon theobromicola is distantly related to species of Cylindrocarpon sensu stricto or Neonectria sensu stricto. It clustered among cylindrocarpon-like species with curved macroconidia, of which some belong to the Neon. mammoidea group. Relatively voluminous cells in sporodochial conidiophores of Cyl. theobromicola resembled those described for Campylocarpon, which is closely related to members of the Neon. mammoidea group including Cyl. theobromicola. Cylindrocarpon pauciseptatum has been isolated from roots of Vitis spp. in South-eastern Europe (Slovenia) as well as New Zealand, where it also occurs on roots of Erica melanthera.

Neonectria liriodendri sp. nov., the main causal agent of black foot disease of grapevines.

Black foot disease is a serious disease of grapevine crops in most areas where vines are grown. Mainly two species of Cylindrocarpon, C. destructans and C. macrodidymum, are associated with this disease. Recent studies have revealed a tremendous molecular variation within the former but only slight molecular variation within the latter, indicating that C. destructans presents a complex of several species The present study elucidates the taxonomic status of C. destructans-like isolates associated with black foot disease of grapevines. Grapevine isolates were studied morphologically, subjected to DNA analyses of their ITS and partial beta-tubulin genes, and were mated in all combinations in vitro. Cylindrocarpon destructans strains isolated from grapevines in Europe and South Africa appeared morphologically and genetically identical, and had identical ITS and partial beta-tubulin gene sequences. Phylogenetic analyses placed these strains in a clade closely related but clearly distinct from other clades with C. destructans-like anamorphs obtained from various herbaceous or woody hosts. Only the ex-type strain of Cylindrocarpon liriodendri had identical sequences to strains isolated from grapevines, and could also not be distinguished by morphological characters. The grapevine isolates are therefore reidentified here as Cylindrocarpon liriodendri. Cylindrocarpn liriodendri formed perithecia in heterothallic conditions and the holomorph of this species is described as Neonectria liriodendri sp. nov. Neonectria liriodendri is genetically distinct from the ex-type strain of Neonectria radicicola, which originated from Cyclamen in Sweden. Both ex-type strains also differ from at least two other clades comprising additional C. destructans-like strains. Many of these strains originated from Panax sp., which is the host of the type of C. destructans. Our phylogenetic analyses indicate that C. destructans is not the anamorph of N. radicicola and that N. liriodendri, N. radicicola and several C. destructans-like taxa may have evolved independently within the same phylogenetic species complex.

DNA phylogeny, morphology and pathogenicity of Botryosphaeria species on grapevines.

Several species of Botr yosphaeria are known to occur on grapevines, causing a wide range of disorders including bud mortality, dieback, brown wood streaking and bunch rot. In this study the 11 Botryosphaeria spp. associated with grapevines growing in various parts of the world, but primarily in South Africa, are distinguished based on morphology, DNA sequences (ITS-1, 5.8S, ITS-2 and EF1-α) and pathological data. Botryosphaeria australis, B. lutea, B. obtusa, B. parva, B. rhodina and a Diplodia sp. are confirmed from grapevines in South Africa, while Diplodia porosum, Fusicoccum viticlavatum and F. vitifusiforme are described as new. Although isolates of B. dothidea and B. stevensii are confirmed from grapevines in Portugal, neither of these species occurred in South Africa, nor were any isolates of B. ribis confirmed from grapevines. All grapevine isolates from Portugal, formerly presumed to be B. ribis, are identified as B. parva based on their EF1-α equence data. From artificial inoculations on grapevine shoots, we conclude that B. australis, B. parva, B. ribis and B. stevensii are more virulent than the other species studied. The Diplodia sp. collected from grapevine canes is morphologically similar but phylogenetically distinct from D. sarmentorum. Diplodia sarmentorum is confirmed as anamorph of Otthia spiraeae, the type species of the genus Otthia (Botryosphaeriaceae). A culture identified as O. spiraeae clustered within Botryosphaeria and thus is regarded as probable synonym. These findings confirm earlier suggestions that the generic concept of Botryosphaeria should be expanded to include genera with septate ascospores and Diplodia anamorphs.

Proactive Control of Petri Disease of Grapevine Through Treatment of Propagation Material.

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.