Dieback and decline pathogens of olive trees in South Africa.

Trunk disease fungal pathogens reduce olive production globally by causing cankers, dieback, and other decline-related symptoms on olive trees. Very few fungi have been reported in association with olive dieback and decline in South Africa. Many of the fungal species reported from symptomatic olive trees in other countries have broad host ranges and are known to occur on other woody host plants in the Western Cape province, the main olive production region of South Africa. This survey investigated the diversity of fungi and symptoms associated with olive dieback and decline in South Africa. Isolations were made from internal wood symptoms of 145 European and 42 wild olive trees sampled in 10 and 9 districts, respectively. A total of 99 taxa were identified among 440 fungal isolates using combinations of morphological and molecular techniques. A new species of Pseudophaeomoniella, P. globosa, had the highest incidence, being recovered from 42.8 % of European and 54.8 % of wild olive samples. This species was recovered from 9 of the 10 districts where European olive trees were sampled and from all districts where wild olive trees were sampled. Members of the Phaeomoniellales (mainly P. globosa) were the most prevalent fungi in five of the seven symptom types considered, the only exceptions being twig dieback, where members of the Botryosphaeriaceae were more common, and soft/white rot where only Basidiomycota were recovered. Several of the species identified are known as pathogens of olives or other woody crops either in South Africa or elsewhere in the world, including species of Neofusicoccum, Phaeoacremonium, and Pleurostoma richardsiae. However, 81 of the 99 taxa identified have not previously been recorded on olive trees and have unknown interactions with this host. These taxa include one new genus and several putative new species, of which four are formally described as Celerioriella umnquma sp. nov., Pseudophaeomoniella globosa sp. nov., Vredendaliella oleae gen. & sp. nov., and Xenocylindrosporium margaritarum sp. nov.

Phaeoacremonium species diversity on woody hosts in the Western Cape Province of South Africa.

Nineteen Phaeoacremonium species are currently known in South Africa. These have been reported from grapevines, fruit trees, fynbos twig litter and arthropods. In other countries some of these Phaeoacremonium species are also known from hosts such as European olive, quince and willow that commonly occur in the Western Cape Province of South Africa, where most South African records of Phaeoacremonium have been made. The aim of this study was to investigate the species diversity and host-range of Phaeoacremonium in the Western Cape Province of South Africa by characterising 156 isolates collected from 29 woody hosts. Phylogenetic analyses of combined actin and beta-tubulin datasets allowed for the identification of 31 species among the 156 isolates, including 13 new species and 3 known species that had not been recorded in South Africa previously. The new Phaeoacremonium species include P. album, P. aureum, P. bibendum, P. gamsii, P. geminum, P. junior, P. longicollarum, P. meliae, P. oleae, P. paululum, P. proliferatum, P. rosicola and P. spadicum. All previous records of P. alvesii in South Africa were re-identified as P. italicum, but both species were recovered during this survey. A total of 35 described Phaeoacremonium species are now known from South Africa, more than double the number reported from any other country. This high diversity reflects the high diversity of indigenous flora of the Cape Floral Region, a biodiversity hotspot mainly situated in the Western Cape Province. Paraphyly and incongruence between individual phylogenies of the actin and beta-tubulin regions complicated species delimitation in some cases indicating that additional phylogenetic markers should be investigated for use in Phaeoacremonium phylogenies to prevent misidentifications and the introduction of vague species boundaries.

In vitro inhibition of Plasmodium falciparum early and late stage gametocyte viability by extracts from eight traditionally used South African plant species.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of plant species, used traditionally to treat malaria, have been extensively investigated for their activity against Plasmodium intraerythrocytic asexual parasites in search of new antimalarial drugs. However, less effort has been directed towards examining their efficacy in blocking transmission. Here, we report the results of the in vitro screening of extracts from eight selected plant species used traditionally to treat malaria in South Africa for activity against Plasmodium falciparum NF54 early and late stage gametocytes. The species used were Khaya anthotheca, Trichilia emetica, Turraea floribunda, Leonotis leonurus, Leonotis leonurus ex Hort, Olea europaea subsp. Africana, Catha edulis and Artemisia afra. AIM OF THE STUDY: To investigate the activities of extracts from plant species traditionally used for malaria treatment against P. falciparum gametocytes. MATERIAL AND METHODS: Air-dried and ground plant leaves were extracted using acetone. Primary two point in vitro phenotypic screens against both early and late stage gametocytes were done at 10 and 20µg/ml followed by full IC50 determination of the most active extracts. Inhibition of gametocyte viability in vitro was assessed using the parasite lactate dehydrogenase (pLDH) assay. RESULTS: Of the eight crude acetone extracts from plant species screened in vitro, four had good activity with over 50-70% inhibition of early and late stage gametocytes' viability at 10 and 20µg/ml, respectively. Artemisia afra (Asteraceae), Trichilia emetica (Meliaceae) and Turraea floribunda (Meliaceae) were additionally highly active against both gametocyte stages with IC50 values of less than 10µg/ml while Leonotis leonurus ex Hort (Lamiaceae) was moderately active (IC50<20µg/ml). The activity of these three highly active plant species was significantly more pronounced on late stage gametocytes compared to early stages. CONCLUSION: This study shows the potential transmission blocking activity of extracts from selected South African medicinal plants and substantiates their traditional use in malaria control that broadly encompasses prevention, treatment and transmission blocking. Further studies are needed to isolate and identify the active principles from the crude extracts of A. afra, T. emetica and T. floribunda, as well as to examine their efficacy towards blocking parasite transmission to mosquitoes.

Trunk Disease Fungi Associated With Diospyros kaki in South Africa.

Persimmon trees with dieback symptoms and cankers were observed in three production areas in Western Cape Province in South Africa. Isolations were made from diseased branches, cankers, and pruning wounds as well as fungal fruiting bodies on dead branches and old pruning wounds. Several trunk disease pathogens were identified based on morphological characteristics and by molecular methods, including Diaporthe eres, D. infecunda, Eutypella citricola, E. microtheca, Phaeoacremonium parasiticum, P. scolyti, P. australiense, P. minimum, Fomitiporia capensis, Fomitiporia sp., Fomitiporella sp., and Inocutis sp., which were isolated from persimmon for the first time in the world. Other first reports from persimmon in South Africa include D. foeniculina, D. ambigua, D. mutila, Diaporthe sp., Neofusicoccum australe, N. parvum, Diplodia seriata, and Eutypa lata. Pathogenicity tests conducted with all species, except the basidiomycetes, confirmed their status as possible persimmon pathogens. This is the first study to determine and identify fungi associated with diseased persimmon in South Africa. The knowledge gained in this study forms the basis for further research to determine the impact of these fungi on persimmon productivity.

Arthropods vector grapevine trunk disease pathogens.

Arthropod-mediated dispersal of pathogens is known in many cropping systems but has never been demonstrated for grapevine trunk disease pathogens. Arthropods from vineyards were screened for the presence of pathogens associated with Petri disease and esca using cultural and molecular techniques. The ability of the most abundant pathogen-carrying species to inoculate healthy grapevine vascular tissues was also determined. Millipedes and ants were allowed to associate with a DsRed- Express-transformed Phaeomoniella chlamydospora, after which they were exposed to freshly pruned healthy grapevines under controlled conditions and wounds were monitored for subsequent infection. In addition, the possibility of millipede excreta, commonly found on pruning wounds in the field, to act as inoculum source was determined. A diverse arthropod fauna was associated with declining grapevines and many of these carried trunk disease pathogens. However, spiders, the ant Crematogaster peringueyi, and the millipede Ommattoiulus moreleti were the most abundant pathogen carriers. The ant and millipede species fed on pruning wound sap and effectively transmitted trunk disease pathogens. Millipede excreta contained viable spores of Phaeomoniella chlamydospora and may serve as an inoculum source. Numerous arthropods, including beneficial predators, are potential vectors of grapevine trunk disease pathogens. Our results highlight the need for an integrated approach, including targeted management of ants and millipedes at the time of pruning, to limit the spread of grapevine trunk diseases.

Collection, in vitro fertilisation and culture of bovine oocytes intravaginally or in a conventional incubator.

The aim of this study was to test the feasibility of intravaginal culture for in vitro fertilisation and production of bovine embryos. Oocytes were collected from five cows, superovulated with ovine follicle stimulatory hormone (oFSH), by using a 7.5 MHz rectal linear array ultrasound transducer. From 124 follicles punctured, the oocyte recovery rate was 48 per cent. Fifty-one oocytes were placed in capsules either in the vagina or in an incubator, and cocultured with sperm for 24 hours followed by coculture with oviductal cells for 48 hours. There was no significant difference between the fertilisation rates or the cleavage rates achieved by the two systems and the mean fertilisation rate was 44 per cent. However, the fertilised oocytes did not develop beyond the eight-cell stage.