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.

Novel mating-type-associated genes and gene fragments in the genomes of Mycosphaerellaceae and Teratosphaeriaceae fungi.

The mating-type (MAT1) locus encodes transcription factors essential for the onset of the sexual cycle in ascomycete fungi. This locus has been characterised in only a few heterothallic, plant pathogenic Mycosphaerellaceae and Teratosphaeriaceae. We used available genome sequences for Mycosphaerellales species to investigate the presence of two unique mating-type-associated features. The accessory MAT1 genes, MAT1-1-10 (MATORF2) and MAT1-2-12 (MATORF1), typically occurred in both MAT idiomorphs of Mycosphaerellaceae species. In contrast, they were associated with only one idiomorph in Teratosphaeriaceae species. In Pseudocercospora, phylogenetic analyses showed that homologs present in different idiomorphs were paralogous and subject to different selective pressures, indicating that their evolution is linked to mating type. In almost half of the investigated Mycosphaerellales genomes, numerous short fragment sequences, almost identical to portions of the MAT1-1-1 and MAT1-2-1 genes, were present in multiple areas outside of the MAT1 locus. Aligned to the MAT1 genes, these sequences resembled an mRNA transcript. Fragment sequences were similar among species groups and occurred at the same genomic positions, implying that monophyletic groups have the same origins of these sequences. Although the functions of the MAT fragment sequences and accessory MAT1 genes remain unknown, both were expressed in the representative Mycosphaerellaceae and Teratosphaeriaceae species that were investigated.

Genetic response to nitrogen starvation in the aggressive Eucalyptus foliar pathogen Teratosphaeria destructans.

Teratosphaeria destructans is one of the most aggressive foliar pathogens of Eucalyptus. The biological factors underpinning T. destructans infections, which include shoot and leaf blight on young trees, have never been interrogated. Thus, the means by which the pathogen modifies its host environment to overcome host defences remain unknown. By applying transcriptome sequencing, the aim of this study was to compare gene expression in a South African isolate of T. destructans grown on nitrogen-deficient and complete media. This made it possible to identify upregulated genes in a nitrogen-starved environment, often linked to the pathogenicity of the fungus. The results support the hypothesis that nitrogen starvation in T. destructans likely mirrors an in planta genetic response. This is because 45% of genes that were highly upregulated under nitrogen starvation have previously been reported to be associated with infection in other pathogen systems. These included several CAZymes, fungal effector proteins, peptidases, kinases, toxins, lipases and proteins associated with detoxification of toxic compounds. Twenty-five secondary metabolites were identified and expressed in both nitrogen-deficient and complete conditions. Additionally, the most highly expressed genes in both growth conditions had pathogenicity-related functions. This study highlights the large number of expressed genes associated with pathogenicity and overcoming plant defences. As such, the generated baseline knowledge regarding pathogenicity and aggressiveness in T. destructans is a valuable reference for future in planta work.

Mating strategy and mating type distribution in six global populations of the Eucalyptus foliar pathogen Teratosphaeria destructans.

Teratosphaeria destructans is an aggressive fungal pathogen causing leaf and shoot blight on young Eucalyptus trees in plantations. The disease occurs across tropical and subtropical regions of South East Asia and has recently been found in South Africa. Asexual structures of the pathogen are produced on infected tissues, but sexual structures have never been observed. The aim of this study was to investigate the reproductive biology of T. destructans by characterising its mating type (MAT1) locus and investigating its potential for sexual recombination. We found that T. destructans has a heterothallic mating system, with either the MAT1-1-1 and MAT1-1-10 genes (MAT1-1 idiomorph) or the MAT1-2-1 and MAT1-2-12 genes (MAT1-2 idiomorph) present in a single individual. With a multiplex PCR assay, it was possible to distinguish the two MAT idiomorphs in several Teratosphaeria species and this approach was applied to six global populations of T. destructans. Although both mating types occurred in the South East Asian populations, a single mating type dominated each population. Isolates from the recent disease outbreak in South Africa comprised only a single mating type. Attempts to induce a sexual cycle in vitro using strains of opposite mating type were not successful. The uneven distribution of mating types in populations of T. destructans and the presence of only an asexual state on infected tissues suggests the absence of or at least a minor role for sexual reproduction where the pathogen occurs on non-native Eucalyptus in plantations.

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.

Nine draft genome sequences of Claviceps purpurea s.lat., including C. arundinis, C. humidiphila, and C. cf. spartinae, pseudomolecules for the pitch canker pathogen Fusarium circinatum, draft genome of Davidsoniella eucalypti, Grosmannia galeiformis, Quambalaria eucalypti, and Teratosphaeria destructans.

This genome announcement includes draft genomes from Claviceps purpurea s.lat., including C. arundinis, C. humidiphila and C. cf. spartinae. The draft genomes of Davidsoniella eucalypti, Quambalaria eucalypti and Teratosphaeria destructans, all three important eucalyptus pathogens, are presented. The insect associate Grosmannia galeiformis is also described. The pine pathogen genome of Fusarium circinatum has been assembled into pseudomolecules, based on additional sequence data and by harnessing the known synteny within the Fusarium fujikuroi species complex. This new assembly of the F. circinatum genome provides 12 pseudomolecules that correspond to the haploid chromosome number of F. circinatum. These are comparable to other chromosomal assemblies within the FFSC and will enable more robust genomic comparisons within this species complex.