Fahimeh Jami

Five New Species of the Botryosphaeriaceae from Acacia karroo in South Africa

Abstract The Botryosphaeriaceae represents an important, cosmopolitan family of latent pathogens infecting woody plants. Recent studies on native trees in southern Africa have revealed an extensive diversity of species of Botryosphaeriaceae, about half of which have not been previously described. This study adds to this growing body of knowledge, by discovering five new species of the Botryosphaeriaceae on Acacia karroo, a commonly occurring native tree in southern Africa. These species were isolated from both healthy and diseased tissues, suggesting they could be latent pathogens. The isolates were compared to other species for which DNA sequence data are available using phylogenetic analyses based on the ITS, TEF-1&agr;, &bgr;-tubulin and LSU gene regions, and characterized based on their morphology. The morphological data were, however, useful to make comparisons with other species found in the same region and on similar hosts. The five new species were described as Diplodia allocellula, Dothiorella dulcispinae, Do. brevicollis, Spencermartinsia pretoriensis and Tiarosporella urbis-rosarum. Evidence emerging from this study suggests that many more species of the Botryosphaeriaceae remain to be discovered in the southern Africa.

Confronting the constraints of morphological taxonomy in the Botryosphaeriales

Identification of fungi and the International Code of Nomenclature underpinning this process, rests strongly on the characterisation of morphological structures. Yet, the value of these characters to define species in many groups has become questionable or even superfluous. This has emerged as DNA-based techniques have increasingly revealed cryptic species and species complexes. This problem is vividly illustrated in the present study where 105 isolates of the Botryosphaeriales were recovered from both healthy and diseased woody tissues of native Acacia spp. in Namibia and South Africa. Thirteen phylogenetically distinct groups were identified based on Internal Transcribed Spacer (ITS) rDNA PCR-RFLP and translation elongation factor 1-α (TEF1-α) sequence data, two loci that are known to be reliable markers to distinguish species in the Botryosphaeriales. Four of these groups could be linked reliably to sequence data for formerly described species, including Botryosphaeria dothidea, Dothiorella dulcispinae, Lasiodiplodia pseudotheobromae and Spencermartinsia viticola. Nine groups, however, could not be linked to any other species known from culture and for which sequence data are available. These groups are, therefore, described as Aplosporella africana, A. papillata, Botryosphaeria auasmontanum, Dothiorella capri-amissi, Do. oblonga, Lasiodiplodia pyriformis, Spencermartinsia rosulata, Sphaeropsis variabilis and an undescribed Neofusicoccum sp. The species described here could not be reliably compared with the thousands of taxa described in these genera from other hosts and regions, where only morphological data are available. Such comparison would be possible only if all previously described taxa are epitypified, which is not a viable objective for the two families, Botryosphaeriaceae and Aplosporellaceae, in the Botryosphaeriales identified here. The extent of diversity of the Botryosphaeriales revealed in this and other recent studies is expected to reflect that of other undersampled regions and hosts, and illustrates the urgency to find more effective ways to describe species in this, and indeed other, groups of fungi.

Families, genera, and species of Botryosphaeriales

Members of Botryosphaeriales are ecologically diverse, but most commonly associated with leaf spots, fruit and root rots, die-back or cankers of diverse woody hosts. Based on morphology and DNA sequence data, the Botryosphaeriales have to date been shown to contain eight families, with an additional two, Endomelanconiopsisaceae (Endomelanconiopsis) and Pseudofusicoccumaceae (Pseudofusicoccum) being newly described in this study. Furthermore, Oblongocollomyces is introduced as new genus, while Spencermartinsia is reduced to synonymy under Dothiorella. Novel species include Diplodia pyri (Pyrus sp., the Netherlands), Diplodia citricarpa (Citrus sp., Iran), Lasiodiplodia vitis (Vitis vinifera, Italy), L. sterculiae (Sterculia oblonga, Germany), Neofusicoccum pistaciarum (Pistacia vera, USA), N. buxi (Buxus sempervirens, France), N. stellenboschiana (Vitis vinifera, South Africa), and Saccharata hawaiiensis (Protea laurifolia, Hawaii). New combinations are also proposed for Camarosporium pistaciae (associated with fruit rot of Pistacia vera) in Neofusicoccum, and Sphaeria gallae (associated with galls of Quercus) in Diplodia. The combination of large subunit of the nuclear ribosomal RNA gene (LSU)-rpb2 proved effective at delineating taxa at family and generic level. Furthermore, rpb2 also added additional resolution for species delimitation, in combination with ITS, tef1 and tub2. In this study we analysed 499 isolates, and produce an expanded phylogenetic backbone for Botryosphaeriales, which will help to delimit novelties at species, genus and family level in future.

Botryosphaeriaceae species overlap on four unrelated, native South African hosts

Botryosphaeriaceae represents an important and diverse family of latent fungal pathogens of woody plants. We address the question of host range of these fungi by sampling leaves and branches of four native South African trees, including Acacia karroo (Fabaceae), Celtis africana (Cannabaceae), Searsia lancea (Anacardiaceae), and Gymnosporia buxifolia (Celastraceae). Two new species of the Botryosphaeriaceae, namely Tiarosporella africana sp. nov. and Aplosporella javeedii sp. nov. were identified, together with five known species, including Neofusicoccum parvum, Neofusicoccum kwambonambiense, Spencermartinsia viticola, Diplodia pseudoseriata, and Botryosphaeria dothidea. Most Botryosphaeriaceae occurred on more than one host. With the exception of S. lancea, which was infected by A. javeedii all the hosts were infected by more than one Botryosphaeriaceae species. Collectively, the results suggest that some intrinsic host factors, possibly combined with local environmental conditions, affect the distribution and co-infectivity of various hosts by the Botryosphaeriaceae. This would counteract the general ability of a species in the Botryosphaeriaceae to infect a broad range of plants. The combination of host and environmental factors might also explain why some Botryosphaeriaceae with apparently broad host ranges, are found on different suites of hosts in different areas of the world.

Greater Botryosphaeriaceae diversity in healthy than associated diseased Acacia karroo tree tissues

Botryosphaeriaceae are common endophytes of trees. Some species are also known to be pathogens. It is, therefore, assumed that endophytic Botryosphaeriaceae are often involved in general die-back diseases. Here we test this assumption in severe branch die-back observed on Acacia karroo trees in the Pretoria area of South Africa. The presence of the Botryosphaeriaceae was compared between healthy and diseased tissue on the same trees. Eight Botryosphaeriaceae species were isolated from die-back and healthy branches. Of these, six species, namely Tiarosporella urbis-rosarum, Diplodia allocellula, Phaeobotryosphaeria variabilis, Dothiorella brevicollis and Neofusicoccum vitifusiforme were obtained from healthy tissues, and only two species, Dothiorella dulcispinae and Spencermartinsia pretoriensis, were exclusively found in die-back branches. Spencermartinsia viticola was found in both tissue types and this fungus was also the most commonly isolated species from both healthy and die-back samples. Results of pathogenicity trials showed highly variable results for the isolated species and that the two species associated only with die-back symptoms, were weakly pathogenic. These results suggest that the Botryosphaeriaceae found in these trees were not directly associated with the die-back symptoms, despite their diversity and common occurrence in these tissues. The situation is different in other tree systems where dominant species, often with wide host ranges, have been shown to be involved in die-back diseases. This indicates the importance of characterizing the unique aspects of each tree disease system.

Botryosphaeria dothidea : a latent pathogen of global importance to woody plant health

Botryosphaeria dothidea is the type species of Botryosphaeria (Botryosphaeriaceae, Botryosphaeriales). Fungi residing in this order are amongst the most widespread and important canker and dieback pathogens of trees worldwide, with B. dothidea one of the most common species on a large number of hosts. Its taxonomic circumscription has undergone substantial change in the past decade, making it difficult to interpret the large volume of literature linked to the name B. dothidea. This pathogen profile synthesizes the current understanding of B. dothidea pertaining to its distribution, host associations and role as a pathogen in managed and natural woody environments. The prolonged latent infection or endophytic phase is of particular importance, as it implies that the fungus can easily pass undetected by quarantine systems in traded living plants, fruits and other plant parts. Infections typically become obvious only under conditions of host stress, when disease symptoms develop. This study also considers the knowledge emerging from the recently sequenced B. dothidea genome, elucidating previously unknown aspects of the species, including mating and host infection strategies. Despite more than 150 years of research on B. dothidea, there is clearly much to be learned regarding this global tree pathogen. This is increasingly important given the stresses imposed on various woody hosts as a result of climate change. TAXONOMY Botryosphaeria dothidea (Moug. ex Fr) Ces. & De Not, 1863. Kingdom Fungi, Phylum Ascomycota, Class Dothideomycetes, Order Botryosphaeriales, Family Botryosphaeriaceae, Genus Botryosphaeria, Species dothidea. HOST RANGE Confirmed on more than 24 host genera, including woody plants, such as Acacia (= Vachellia), Eucalyptus, Vitis and Pistachio. DISEASE SYMPTOMS Associated with twig, branch and stem cankers, tip and branch dieback, fruit rot, blue stain and plant death. USEFUL WEBSITES The Botryosphaeria site for detailed morphological descriptions (http://www.crem.fct.unl.pt/botryosphaeria_site/); Systematic Mycology and Microbiology Laboratory Fungal Database for all literature and associated hosts (https://nt.ars-grin.gov/fungaldatabases/); TreeBASE link for the combined ITS and TEF-1α tree (http://purl.org/phylo/treebase/phylows/study/TB2:S18906); DOE Joint Genome Institute, JGI Mycocosm for the Botryosphaeria dothidea genome (http://genome.jgi.doe.gov/Botdo1_1/Botdo1_1.home.html).

Botryosphaeriaceae associated with the die-back of ornamental trees in the Western Balkans

Extensive die-back and mortality of various ornamental trees and shrubs has been observed in parts of the Western Balkans region during the past decade. The disease symptoms have been typical of those caused by pathogens residing in the Botryosphaeriaceae. The aims of this study were to isolate and characterize Botryosphaeriaceae species associated with diseased ornamental trees in Serbia, Montenegro, Bosnia and Herzegovina. Isolates were initially characterized based on the DNA sequence data for the internal transcribed spacer rDNA and six major clades were identified. Representative isolates from each clade were further characterized using DNA sequence data for the translation elongation factor 1-alpha, β-tubulin-2 and large subunit rRNA gene regions, as well as the morphology of the asexual morphs. Ten species of the Botryosphaeriaceae were identified of which eight, i.e., Dothiorella sarmentorum, Neofusicoccum parvum, Botryosphaeria dothidea, Phaeobotryon cupressi, Sphaeropsis visci, Diplodia seriata, D. sapinea and D. mutila were known taxa. The remaining two species could be identified only as Dothiorella spp. Dichomera syn-asexual morphs of D. sapinea, Dothiorella sp. 2 and B. dothidea, as well as unique morphological characters for a number of the known species are described. Based on host plants and geographic distribution, the majority of Botryosphaeriaceae species found represent new records. The results of this study contribute to our knowledge of the distribution, host associations and impacts of these fungi on trees in urban environments.

Diversity in the Botryosphaeriales: Looking back, looking forward

The Botryosphaeriales are amongst the most widespread, common and important fungal pathogens of woody plants. Many are also known to exist as endophytes in healthy plant tissues. This special issue highlights a number of key themes in the study of this group of fungi. In particular, there have been dramatic taxonomic changes over the past decade; from one family to nine (including two in this special issue) and from 10 to 33 genera known from culture. It is also clear from many studies that neither morphology nor single locus sequence data are sufficient to define taxa. This problem is exacerbated by the increasing recognition of cryptic species and hybrids (as highlighted for the first time in this special issue). It is futile that management strategies, including quarantine, continue to rely on outdated taxonomic definitions and identification tools. This is especially true in light of growing evidence that many species continue to be moved globally as endophytes in plants and plant products. A well defined natural classification and an extensive collection of tools to study the Botryosphaeriaceae, including a growing number of genomes, now provide a springboard for a much deeper exploration of their biology, biogeography and host associations.

Diversity of tree-infecting Botryosphaeriales on native and non-native trees in South Africa and Namibia

The Botryosphaeriales includes serious plant pathogens with a broad host and geographic distribution globally. In South Africa and Namibia, these fungi include important pathogens of native and non-native woody plants, and have consequently been studied extensively. Here we synthesize the information from the previous studies, particularly in the last decade, that report 62 species in the Botryosphaeriales from 66 hosts across South Africa and Namibia. Of these, 52 species have been reported from native hosts, 17 are from non-native hosts and twelve of these species occur on both native and non-native trees in the region. Much of the diversity of the Botryosphaeriales can be ascribed to native species that have fairly limited host and geographic ranges. Neofusicoccum parvum is amongst the most common species on both native and non-native hosts and it is thought to be native to the region. In contrast, Botryosphaeria dothidea, which is certainly an introduced species, is also widespread, and is very common on both native and non-native plants. Overall this synthesis underscores the growing understanding of the diversity of an important group of tree pathogens, their apparently common global spread as latent agents of disease, as well as their apparently common movement between commercial and native ecosystems.

Australian cultures of Botryosphaeriaceae held in Queensland and Victoria plant pathology herbaria revisited

The Botryosphaeriaceae is one of the most widespread and cosmopolitan endophytic group of fungi. However, the species of this group can cause severe disease when the hosts are under stressful conditions. The aim of this study was to identify living cultures from the Botryosphaeriaceae family preserved in the Queensland and Victorian Plant Pathology Herbaria using DNA sequence analyses. The 51 isolates were collected between 1971 and 2017, from 35 different host genera, with the dominant host genera being Mangifera (11 isolates), Acacia (10), and Persea (5). Multilocus sequence analyses resulted in the re-identification of 41 isolates to the genera Botryosphaeria (2 isolates), Diplodia (4), Dothiorella (1), Lasiodiplodia (19), and Neofusicoccum (15), as well as some that belonged to genera outside of the Botryosphaeriaceae (10). New records for Australia were Botryosphaeria sinensis, Diplodia alatafructa, Lasiodiplodia gonubiensis, Neofusicoccum cryptoaustrale, and N. mangroviorum. These were identified as a result of a workshop organised by the Subcommittee on Plant Health Diagnostics. The results of this study provide the fundamental information regarding the diversity of Botryosphaeriaceae species present in Australian.