Potential drug-drug interactions with phentermine among long-term phentermine consumers: A retrospective analysis.

BACKGROUND: Phentermine is an internationally recognised amphetamine derivative with significant appetite-suppressing properties. The drug is indicated for the short-term management of obesity, as the long-term (LT) use of phentermine may potentially be associated with severe cardiovascular side-effects, abuse and dependence. The LT use hereinafter describes periods exceeding 12 consecutive weeks. This use may also be associated with potential drug-drug interactions (PDDIs), which may result in adverse drug reactions (ADRs). The literature reports that phentermine is often prescribed LT and for several other off-label indications, increasing the risk for individuals to experience adverse drug events (ADEs) and drug-drug interactions (DDIs). There are, to our knowledge, no South African (SA) studies investigating the prevalence of co-prescribing LT phentermine with drugs that may potentially cause DDIs. OBJECTIVE: To determine the prevalence of mild, moderate and severe DDIs with phentermine use when the duration of therapy in private healthcare exceeded 12 consecutive weeks. METHODS: A cross-sectional drug utilisation review (DUR) was done by using data obtained from a SA pharmacy benefit management (PBM) company's database. Retrospective data of medicine claims for phentermine, from 1  January 2015 to 31  December 2019, were extracted for analysis. The number of days phentermine was supplied was used to identify the study population, in other words, those patients who received the drug LT. A drug interaction checker (Drugs.com) was used to identify potential mild, moderate and severe DDIs when using phentermine and co-prescribed drugs concurrently. RESULTS: A total of 889 patients received phentermine LT. The top 20 drugs identified as being frequently co-prescribed in this study population demonstrated no mild PDDI, 15 (75%) moderate PDDIs and 5 (25%) severe PDDIs. The most common co-prescribed drug in the moderate group was dextromethorphan (n=282, 31.72%) and the least co-prescribed was formoterol (n=52, 5.85%). Among the drug group 'severe PDDIs', tramadol (n=416, 46.79%) was most frequently prescribed, whereas phenylpropanolamine (n=69, 7.76%) was the least prescribed to patients in this group. CONCLUSION: There are patients who receive LT phentermine therapy despite the potential severe consequences that may result. These patients may receive concomitant therapy with phentermine and other pharmaceutical constituents, which may potentially cause DDIs, more specifically, moderate and severe DDIs. As such, these patients are not only confronted with the consequences of DDIs but are also at risk to experience ADRs as the residual effect of PDDIs.

Genera of phytopathogenic fungi: GOPHY 4.

This paper is the fourth contribution in the Genera of Phytopathogenic Fungi (GOPHY) series. The series provides morphological descriptions and information about the pathology, distribution, hosts and disease symptoms, as well as DNA barcodes for the taxa covered. Moreover, 12 whole-genome sequences for the type or new species in the treated genera are provided. The fourth paper in the GOPHY series covers 19 genera of phytopathogenic fungi and their relatives, including Ascochyta, Cadophora, Celoporthe, Cercospora, Coleophoma, Cytospora, Dendrostoma, Didymella, Endothia, Heterophaeomoniella, Leptosphaerulina, Melampsora, Nigrospora, Pezicula, Phaeomoniella, Pseudocercospora, Pteridopassalora, Zymoseptoria, and one genus of oomycetes, Phytophthora. This study includes two new genera, 30 new species, five new combinations, and 43 typifications of older names. Taxonomic novelties: New genera: Heterophaeomoniella L. Mostert, C.F.J. Spies, Halleen & Gramaje, Pteridopassalora C. Nakash. & Crous; New species: Ascochyta flava Qian Chen & L. Cai, Cadophora domestica L. Mostert, R. van der Merwe, Halleen & Gramaje, Cadophora rotunda L. Mostert, R. van der Merwe, Halleen & Gramaje, Cadophora vinacea J.R. Úrbez-Torres, D.T. O'Gorman & Gramaje, Cadophora vivarii L. Mostert, Havenga, Halleen & Gramaje, Celoporthe foliorum H. Suzuki, Marinc. & M.J. Wingf., Cercospora alyssopsidis M. Bakhshi, Zare & Crous, Dendrostoma elaeocarpi C.M. Tian & Q. Yang, Didymella chlamydospora Qian Chen & L. Cai, Didymella gei Qian Chen & L. Cai, Didymella ligulariae Qian Chen & L. Cai, Didymella qilianensis Qian Chen & L. Cai, Didymella uniseptata Qian Chen & L. Cai, Endothia cerciana W. Wang. & S.F. Chen, Leptosphaerulina miscanthi Qian Chen & L. Cai, Nigrospora covidalis M. Raza, Qian Chen & L. Cai, Nigrospora globospora M. Raza, Qian Chen & L. Cai, Nigrospora philosophiae-doctoris M. Raza, Qian Chen & L. Cai, Phytophthora transitoria I. Milenkovic, T. Májek & T. Jung, Phytophthora panamensis T. Jung, Y. Balci, K. Broders & I. Milenkovic, Phytophthora variabilis T. Jung, M. Horta Jung & I. Milenkovic, Pseudocercospora delonicicola C. Nakash., L. Suhaizan & I. Nurul Faziha, Pseudocercospora farfugii C. Nakash., I. Araki, & Ai Ito, Pseudocercospora hardenbergiae Crous & C. Nakash., Pseudocercospora kenyirana C. Nakash., L. Suhaizan & I. Nurul Faziha, Pseudocercospora perrottetiae Crous, C. Nakash. & C.Y. Chen, Pseudocercospora platyceriicola C. Nakash., Y. Hatt, L. Suhaizan & I. Nurul Faziha, Pseudocercospora stemonicola C. Nakash., Y. Hatt., L. Suhaizan & I. Nurul Faziha, Pseudocercospora terengganuensis C. Nakash., Y. Hatt., L. Suhaizan & I. Nurul Faziha, Pseudocercospora xenopunicae Crous & C. Nakash.; New combinations: Heterophaeomoniella pinifoliorum (Hyang B. Lee et al.) L. Mostert, C.F.J. Spies, Halleen & Gramaje, Pseudocercospora pruni-grayanae (Sawada) C. Nakash. & Motohashi., Pseudocercospora togashiana (K. Ito & Tak. Kobay.) C. Nakash. & Tak. Kobay., Pteridopassalora nephrolepidicola (Crous & R.G. Shivas) C. Nakash. & Crous, Pteridopassalora lygodii (Goh & W.H. Hsieh) C. Nakash. & Crous; Typification: Epitypification: Botrytis infestans Mont., Cercospora abeliae Katsuki, Cercospora ceratoniae Pat. & Trab., Cercospora cladrastidis Jacz., Cercospora cryptomeriicola Sawada, Cercospora dalbergiae S.H. Sun, Cercospora ebulicola W. Yamam., Cercospora formosana W. Yamam., Cercospora fukuii W. Yamam., Cercospora glochidionis Sawada, Cercospora ixorana J.M. Yen & Lim, Cercospora liquidambaricola J.M. Yen, Cercospora pancratii Ellis & Everh., Cercospora pini-densiflorae Hori & Nambu, Cercospora profusa Syd. & P. Syd., Cercospora pyracanthae Katsuki, Cercospora horiana Togashi & Katsuki, Cercospora tabernaemontanae Syd. & P. Syd., Cercospora trinidadensis F. Stevens & Solheim, Melampsora laricis-urbanianae Tak. Matsumoto, Melampsora salicis-cupularis Wang, Phaeoisariopsis pruni-grayanae Sawada, Pseudocercospora angiopteridis Goh & W.H. Hsieh, Pseudocercospora basitruncata Crous, Pseudocercospora boehmeriigena U. Braun, Pseudocercospora coprosmae U. Braun & C.F. Hill, Pseudocercospora cratevicola C. Nakash. & U. Braun, Pseudocercospora cymbidiicola U. Braun & C.F. Hill, Pseudocercospora dodonaeae Boesew., Pseudocercospora euphorbiacearum U. Braun, Pseudocercospora lygodii Goh & W.H. Hsieh, Pseudocercospora metrosideri U. Braun, Pseudocercospora paraexosporioides C. Nakash. & U. Braun, Pseudocercospora symploci Katsuki & Tak. Kobay. ex U. Braun & Crous, Septogloeum punctatum Wakef.; Neotypification: Cercospora aleuritis I. Miyake; Lectotypification: Cercospora dalbergiae S.H. Sun, Cercospora formosana W. Yamam., Cercospora fukuii W. Yamam., Cercospora glochidionis Sawada, Cercospora profusa Syd. & P. Syd., Melampsora laricis-urbanianae Tak. Matsumoto, Phaeoisariopsis pruni-grayanae Sawada, Pseudocercospora symploci Katsuki & Tak. Kobay. ex U. Braun & Crous. Citation: Chen Q, Bakhshi M, Balci Y, Broders KD, Cheewangkoon R, Chen SF, Fan XL, Gramaje D, Halleen F, Horta Jung M, Jiang N, Jung T, Májek T, Marincowitz S, Milenkovic T, Mostert L, Nakashima C, Nurul Faziha I, Pan M, Raza M, Scanu B, Spies CFJ, Suhaizan L, Suzuki H, Tian CM, Tomsovský M, Úrbez-Torres JR, Wang W, Wingfield BD, Wingfield MJ, Yang Q, Yang X, Zare R, Zhao P, Groenewald JZ, Cai L, Crous PW (2022). Genera of phytopathogenic fungi: GOPHY 4. Studies in Mycology 101: 417-564. doi: 10.3114/sim.2022.101.06.

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.

Genera of phytopathogenic fungi: GOPHY 2.

This paper represents the second contribution in the Genera of Phytopathogenic Fungi (GOPHY) series. The series provides morphological descriptions and information regarding the pathology, distribution, hosts and disease symptoms for the treated genera. In addition, primary and secondary DNA barcodes for the currently accepted species are included. This second paper in the GOPHY series treats 20 genera of phytopathogenic fungi and their relatives including: Allantophomopsiella, Apoharknessia, Cylindrocladiella, Diaporthe, Dichotomophthora, Gaeumannomyces, Harknessia, Huntiella, Macgarvieomyces, Metulocladosporiella, Microdochium, Oculimacula, Paraphoma, Phaeoacremonium, Phyllosticta, Proxypiricularia, Pyricularia, Stenocarpella, Utrechtiana and Wojnowiciella. This study includes the new genus Pyriculariomyces, 20 new species, five new combinations, and six typifications for older names.

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.

Phylogeny and taxonomy of the scab and spot anthracnose fungus Elsinoë (Myriangiales, Dothideomycetes).

Species of Elsinoë are phytopathogens causing scab and spot anthracnose on many plants, including some economically important crops such as avocado, citrus, grapevines, and ornamentals such as poinsettias, field crops and woody hosts. Disease symptoms are often easily recognisable, and referred to as signature-bearing diseases, for the cork-like appearance of older infected tissues with scab-like appearance. In some Elsinoë-host associations the resulting symptoms are better described as spot anthracnose. Additionally the infected plants may also show mild to severe distortions of infected organs. Isolation of Elsinoë in pure culture can be very challenging and examination of specimens collected in the field is often frustrating because of the lack of fertile structures. Current criteria for species recognition and host specificity in Elsinoë are unclear due to overlapping morphological characteristics, and the lack of molecular and pathogenicity data. In the present study we revised the taxonomy of Elsinoë based on DNA sequence and morphological data derived from 119 isolates, representing 67 host genera from 17 countries, including 64 ex-type cultures. Combined analyses of ITS, LSU, rpb2 and TEF1-α DNA sequence data were used to reconstruct the backbone phylogeny of the genus Elsinoë. Based on the single nomenclature for fungi, 26 new combinations are proposed in Elsinoë for species that were originally described in Sphaceloma. A total of 13 species are epitypified with notes on their taxonomy and phylogeny. A further eight new species are introduced, leading to a total of 75 Elsinoë species supported by molecular data in the present study. For the most part species of Elsinoë appear to be host specific, although the majority of the species treated are known only from a few isolates, and further collections and pathogenicity studies will be required to reconfirm this conclusion.

Response of Vitis vinifera cell cultures to Eutypa lata and Trichoderma atroviride culture filtrates: expression of defence-related genes and phenotypes.

Cell suspension cultures of Vitis vinifera cv. Dauphine berries were used to study the response to the vascular pathogen, Eutypa lata, in comparison with a biological control agent, Trichoderma atroviride, that was previously shown to be effective in pruning wound protection. The expression of genes coding for enzymes of the phenylpropanoid pathway and pathogenesis-related (PR) proteins was profiled over a 48-h period using quantitative reverse transcriptase PCR. The cell cultures responded to elicitors of both fungi with a hypersensitive-like response that lead to a decrease in cell viability. Similar genes were triggered by both the pathogen and biocontrol agent, but the timing patterns and magnitude of expression was dependent on the specific fungal elicitor. Culture filtrates of both fungi caused upregulation of phenylalanine ammonia-lyase (PAL), 4-coumaroyl Co-A ligase (CCo-A) and stilbene synthase (STS), and a downregulation of chalcone synthase (CHS) genes. The pathogen filtrate caused a biphasic pattern in the upregulation of PAL and STS genes which was not observed in cells treated with filtrates of the biocontrol agent. Analytical assays showed significantly higher total phenolic content and chitinolytic enzyme activity in the cell cultures treated with the T. atroviride filtrate compared to the pathogen filtrate. These results corresponded well to the higher expression of PAL and chitinase class IV genes. The response of the cell cultures to T. atroviride filtrate provides support for the notion that the wound protection by the biocontrol agent at least partially relies on the induction of grapevine resistance mechanisms.

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.

First Report of Togninia minima Perithecia on Esca- and Petri-Diseased Grapevines in South Africa.

Esca and petri diseases are important grapevine trunk diseases in South Africa and most other grape-producing countries. The causal pathogens are Phaeomoniella chlamydospora and several species of Phaeoacremonium. In total, 25 species of Phaeoacremonium have been isolated from grapevines of which seven species have been linked to Togninia teleomorphs obtained through in vitro mating studies (3). Of these species, only perithecia of T. minima, T. fraxinopennsylvanica, and T. viticola have been found on grapevines in California (1,2,4). T. minima is heterothallic, and although both mating types are present in South African vineyards, perithecia have never been observed (3). In the current study, grapevine cordons and trunks were collected from vineyards and rootstock mother vines within Western Cape Province for examination in the laboratory under a dissecting microscope. The grapevines displayed general decline symptoms, including reduced vegetative growth, dead or dying shoots and cordons, as well as internal vascular streaking and/or a red/black/brown margin next to decayed wood typically associated with esca and petri disease. Rootstock mother vines were apparently healthy, although many old, cracked pruning wounds were visible. Togninia-like perithecia with distinctive long necks were found along the wood crevices, often on old pruning wounds. The perithecia were removed and placed on microscope slides with sterile water. Structures were measured and slides were washed with 500 µl of sterile water onto potato dextrose agar amended with chloramphenicol (250 mg/liter). Ascospores were allowed to germinate overnight to obtain single ascospore colonies. Perithecia were found on cultivars Muscat d' Alexandrie and Pinotage (Vitis vinifera) at Stellenbosch in May 2011 and on Ramsey (V. champinii) rootstock mother vines at Slanghoek in June 2012. Perithecia were globose to subglobose, black, and often embedded in the wood tissue but also present on the surface of the wood. The length of the necks was 250 to 300 × 47.5 to 55 µm. The asci were hyaline and ranged from 16 to 25 × 3.5 to 5 µm. Ascospores were hyaline, ellipsoid, and ranged from 5 to 6 × 1.5 to 2 µm. These measurements were similar to those reported by Mostert et al. (3) and Rooney et al. (4). Colony growth was typical of T. minima. DNA was extracted from the colonies and the partial betatubulin gene was amplified and sequenced using the primers T1 and Bt2b. Sequences were deposited into GenBank (JX962864 to 67). Based on a megablast search of the NCBI's GenBank nucleotide database, 100% similarity was found with other T. minima sequences (JQ691670.1, HQ605018.1, HQ605014.1; identities = 647/647 [100%], gaps = 0/647 [0%]). To our knowledge, this is the first report on the occurrence of T. minima perithecia on grapevines in Western Cape Province of South Africa. The removal of dead spurs and cordons will be instrumental in lowering the inoculum originating from perithecia, especially in rootstock mother blocks where no control strategies are applied for petri disease or esca. Spore trapping studies are currently in progress to study spore release patterns in order to determine whether pruning wounds are at risk during traditional pruning periods. References: (1) A. Eskalen et al. Plant Dis. 89:528, 2005. (2) A. Eskalen et al. Plant Dis. 89:686, 2005. (3) L. Mostert et al. Stud. Mycol. 54:1, 2006. (4) S. Rooney-Latham et al. Plant Dis. 89:867, 2005.

Fungal trunk pathogens associated with wood decay of almond trees on Mallorca (Spain).

Severe decline of almond trees has recently been observed in several orchards on the island of Mallorca (Balearic Islands, western Mediterranean Sea). However, the identity of the causal agents has not yet been investigated. Between August 2008 and June 2010, wood samples from branches of almond trees showing internal necroses and brown to black vascular streaking were collected in the Llevant region on the island of Mallorca. Several fungal species were subsequently isolated from the margin between healthy and symptomatic tissue. Five species of Botryosphaeriaceae (namely Botryosphaeria dothidea, Diplodia olivarum, D. seriata, Neofusicoccum australe and N. parvum), Eutypa lata, Phaeoacremonium iranianum and Phomopsis amygdali were identified based on morphology, culture characteristics and DNA sequence comparisons. Neofusicoccum parvum was the dominant species, followed by E. lata, D. olivarum and N. australe. First reports from almond include D. olivarum and Pm. iranianum. Two species are newly described, namely Collophora hispanica sp. nov. and Phaeoacremonium amygdalinum sp. nov.

First Report of Leaf Rust of Blueberry Caused by Thekopsora minima on Vaccinium corymbosum in the Western Cape, South Africa.

Southern highbush blueberry plants (Vaccinium corymbosum interspecific hybrids) showing rust-like symptoms were observed in July 2006 in Porterville in the Western Cape (WC), South Africa. Diseased plants were also found in Villiersdorp and George in the WC in 2007. In 2008, symptoms were observed in George, and in 2009, in all the previous reported areas. Cvs. Bluecrisp, Emerald, Jewel, Sharpblue, and Star were infected. Reddish-to-brown spots appeared on the adaxial surface of leaves and developed into yellow-to-orange erumpent uredinia with pulverulent urediniospores. Uredinia were hypophyllous, dome shaped, 113 to 750 µm wide, and occasionally coalescing. Urediniospores were broadly obovate, sometimes ellipsoidal or pyriform, with yellowish orange content, and measured 19 to 27 × 12 to 20 µm (average 24 × 15 µm, n = 30). Spore walls were echinulate, hyaline, 1 to 1.5 µm thick, and with obscure germ pores. No telia or teliospores were observed. Voucher specimens were lodged in the South African National Fungus Collection in Pretoria (PREM 60245). The isolate was initially identified as Thekopsora minima P. Syd. & Syd., based primarily on the absence of conspicuous ostiolar cells characteristic of Naohidemyces spp. (3). Genomic DNA was extracted from urediniospores. Approximately 1,400 bp were amplified spanning the 5.8S, ITS2, and 28S large subunit of the ribosomal DNA (1). The sequence (GU355675) shared 96% (907 of 942 bp; GenBank AF522180) and 94% (1,014 of 1,047 bp; GenBank DQ354563) similarities in the 28S portion, respectively, to those of Naohidemyces vaccinii (Wint.) Sato, Katsuya et Y. Hiratsuka and Pucciniastrum geoppertianum (Kuehn) Kleb, two of the three known rust species of blueberry (2). Although no sequences of T. minima were available for direct comparison, phylogenetic analyses of the 28S region strongly supported the South African blueberry rust as congeneric with T. guttata (J. Schröt.) P. Syd. & Syd. (GenBank AF426231) and T. symphyti (Bubák) Berndt (GenBank AF26230) (data not shown). Four 6-month-old cv. Sharpblue plants were inoculated with a suspension (approximate final concentration of 1 × 105 spores per ml) of fresh urediniospores in a water solution with 0.05% Tween 20. After incubation at 20°C for 48 h under continuous fluorescent lighting, the plants were grown in a glasshouse (18/25°C night/day temperatures). Identical uredinia and symptoms developed approximately 3 weeks after inoculation on the inoculated plants, but not on two control plants of cv. Sharpblue sprayed with distilled water and kept at the same conditions. The alternate host hemlock (Tsuga spp.) is not endemic to South Africa and not sold as an ornamental plant according to a large conifer nursery. Hosts of T. minima include Gaylussacia baccata, G. frondosa, Lyonia neziki, Menziesia pilosa, Rhododendron canadense, R. canescens, R. lutescens R. ponticum, R. prunifolium, R. viscosum, V. angustifolium var. laevifolium, V. corumbosum, and V. erythrocarpon (3). Visual inspection of possible hosts in the gardens in close proximity of Vaccinium production areas did not show any rust symptoms. To our knowledge, this is the first report of T. minima on blueberries outside of Asia and the United States (2). References: (1) M. C. Aime. Mycoscience 47:112, 2006. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Botany and Mycology Laboratory. Online publication. USDA-ARS, 2009. (3) S. Sato et al. Trans. Mycol. Soc. Jpn. 34:47, 1993.

Phytophthora taxa associated with cultivated Agathosma, with emphasis on the P. citricola complex and P. capensis sp. nov.

Agathosma species, which are indigenous to South Africa, are also cultivated for commercial use. Recently growers experienced severe plant loss, and symptoms shown by affected plants suggested that a soilborne disease could be the cause of death. A number of Phytophthora taxa were isolated from diseased plants, and this paper reports their identity, mating type, and pathogenicity to young Agathosma plants. Using morphological and sequence data seven Phytophthora taxa were identified: the A1 mating type of P. cinnamomi var. cinnamomi, P. cinnamomi var. parvispora and P. cryptogea, the A2 mating type of P. drechsleri and P. nicotianae, and two homothallic taxa from the P. citricola complex. The identity of isolates in the P. citricola complex was resolved using reference isolates of P. citricola CIT groups 1 to 5 sensu Oudemans et al. (1994) along with multi-locus phylogenies (three nuclear and two mitochondrial regions), isozyme analyses, morphological characteristics and temperature-growth studies. These analyses revealed the isolates from Agathosma to include P. multivora and a putative novel species, P. taxon emzansi. Furthermore, among the P. citricola reference isolates the presence of a new species was revealed, described here as P. capensis. Findings of our study, along with some recent other studies, have contributed to resolving some of the species complexity within the P. citricola complex, resulting in the identification of a number of phylogenetically distinct taxa. The pathogenicity of representative isolates of the taxa from Agathosma was tested on A. betulina seedlings. The putative novel species, P. taxon emzansi, and P. cinnamomi var. parvispora were non-pathogenic, whereas the other species were pathogenic to this host.

First Report of Phaeoacremonium inflatipes, P. iranianum, and P. sicilianum Causing Petri Disease of Grapevine in Spain.

In 2008, four isolates of Phaeoacremonium, morphologically and genetically different from known Phaeoacremonium spp. in Spain, were isolated from rootstocks of young grapevine (Vitis vinifera) plants showing Petri disease symptoms including low vigor, reduced foliage, and dark streaking of the xylem in Badajoz Province (western Spain; cv. Syrah on SO4 rootstock), Tarragona Province (eastern Spain; cv. Garnacha on 161 49 C rootstock), and Balearic Islands (eastern Spain; cv. Tempranillo on Rupestris de Lot rootstock). Single-conidial isolates were obtained and grown on potato dextrose agar (PDA) and malt extract agar (MEA) at 25°C for 2 to 3 weeks in the dark until colonies sporulated (3). Identification was based on morphological characteristics (1-3). Phaeoacremonium inflatipes W. Gams, Crous & M. J. Wingf. and P. iranianum L. Mostert, Gräf., W. Gams & Crous were detected in Badajoz Province and P. sicilianum Essakhi, Mugnai, Surico & Crous in Tarragona Province and Balearic Islands. Colonies of P. inflatipes were gray on PDA and gray-brown on MEA. Conidiophores were branched, 15 to 37 (mean 25) µm long. Conidia were hyaline, oblong-ellipsoidal or obovoid, 3 to 5.5 (mean 4) µm long, and 1.2 to 1.9 (mean 1.6) µm wide. Colonies of P. iranianum were brownish gray on PDA and pale brown on MEA. Conidiophores were unbranched and 18 to 47.5 (mean 29) µm long. Conidia were hyaline, oblong-ellipsoidal, 3 to 5 (mean 4) µm long, and 1 to 1.8 (mean 1.5) µm wide. Colonies of P. sicilianum were pale brown on PDA and brown to pale orange on MEA. Conidiophores were branched and 13 to 55 (mean 32.5) µm long. Conidia were hyaline, allantoid, 3 to 8.5 (mean 6) µm long, and 1.5 to 2 (mean 1.8) µm wide. Identity of isolates Pin-2, Pir-4, Psi-1, and Psi-2 was confirmed by sequencing a fragment of the beta-tubulin gene with primers T1 and Bt2b (P. inflatipes, isolate Pin-2: GenBank Accession No. FJ872407, 100% similarity to Accession No. AY579323; P. iranianum, isolate Pir-4: GenBank Accession No. FJ872406, 99% similarity to Accession No. EU128077; P. sicilianum isolates Psi-1 and Psi-2: GenBank Accession Nos. FJ872408 and No. FJ872409, 100% similarity to Accession No. EU863489). Pathogenicity tests were conducted using Pin-2, Pir-4, and Psi-1 isolates. One-year-old callused and rooted cuttings of 110 R rootstock cultivated in sterile peat were wounded at the uppermost internode with an 8-mm cork borer. An 8-mm mycelium plug from a 2-week-old culture was placed into the wound. Wounds were wrapped with Parafilm. Ten cuttings per fungal isolate were used. Ten control plants were inoculated with 8-mm noncolonized PDA plugs. Plants were maintained in a greenhouse at 25°C. Within 2 months, all Phaeoacremonium-inoculated cuttings exhibited shoots with poor growth, small leaves, short internodes, and black streaks in the xylem. The mean shoot weight per plant was 1.8 g in P. inflatipes-inoculated plants, 1.9 g in P. iranianum-inoculated plants, and 1.6 g in P. sicilianum-inoculated plants, all lower than the control treatment (6.8 g). Control plants did not show any symptoms. All fungal species were reisolated from wood of all inoculated cuttings, completing Koch's postulates. Their identity was confirmed with the methods described above. To our knowledge, this is the first report of P. inflatipes, P. iranianum, and P. sicilianum causing Petri disease in Spain. References: (1) P. W. Crous et al. Mycologia 88:786, 1996. (2) S. Essakhi et al. Persoonia 21:119, 2008. (3) L. Mostert et al. Stud. Mycol. 54:1, 2006.

Host specificity and speciation of Mycosphaerella and Teratosphaeria species associated with leaf spots of Proteaceae.

Species of Mycosphaerella and Teratosphaeria represent important foliicolous pathogens of Proteaceae. Presently approximately 40 members of these genera (incl. anamorphs) have been recorded from Proteaceae, though the majority are not known from culture, and have never been subjected to DNA sequence analysis. During the course of this study, epitypes were designated for several important species, namely Batcheloromyces leucadendri, B. proteae, Catenulostroma macowanii, Mycosphaerella marksii, Teratosphaeria bellula, T. jonkershoekensis, T. parva, and T. proteae-arboreae. Several species were also newly described, namely Batcheloromyces sedgefieldii, Catenulostroma wingfieldii, Dissoconium proteae, Teratosphaeria persoonii, T. knoxdavesii, and T. marasasii. Although accepted as being highly host specific, some species were shown to have wider host ranges, such as M. communis (Eucalyptus, Protea), M. konae (Leucospermum, Eucalyptus), M. marksii (Eucalyptus, Leucadendron), T. associata (Eucalyptus,Protea), and T. parva (Eucalyptus, Protea), which in most cases were found to co-occur with other species of Mycosphaerella or Teratosphaeria on Proteaceae. Furthermore, earlier records of T. jonkershoekensis on Proteaceae in Australia were shown to be representative of two recently described species, T. associata and T. maxii. A phenomenon of underdeveloped, or micro-ascospores was also newly observed in asci of T. maculiformis and T. proteae-arboreae. The exact purpose of asci with two distinct types of ascospores remains to be clarified, as both types were observed to germinate on agar.

Novel Phaeoacremonium species associated with necrotic wood of Prunus trees.

The genus Phaeoacremonium is associated with opportunistic human infections, as well as stunted growth and die-back of various woody hosts, especially grapevines. In this study, Phaeoacremonium species were isolated from necrotic woody tissue of Prunus spp. (plum, peach, nectarine and apricot) from different stone fruit growing areas in South Africa. Morphological and cultural characteristics as well as DNA sequence data (5.8S rDNA, ITS1, ITS2, beta-tubulin, actin and 18S rDNA) were used to identify known, and describe novel species. From the total number of wood samples collected (257), 42 Phaeoacremonium isolates were obtained, from which 14 species were identified. Phaeoacremonium scolyti was most frequently isolated, and present on all Prunus species sampled, followed by Togninia minima (anamorph: Pm. aleophilum) and Pm. australiense. Almost all taxa isolated represent new records on Prunus. Furthermore, Pm. australiense,Pm. iranianum, T. fraxinopennsylvanica and Pm. griseorubrum represent new records for South Africa, while Pm. griseorubrum, hitherto only known from humans, is newly reported from a plant host. Five species are newly described, two of which produce a Togninia sexual state. Togninia africana, T. griseo-olivacea and Pm. pallidum are newly described from Prunus armeniaca, while Pm. prunicolum and Pm. fuscum are described from Prunus salicina.

First Report of Daylily Rust Caused by Puccinia hemerocallidis in the Western Cape Province of South Africa.

Rust symptoms were first observed on daylily plants (Hemerocallis spp.) during February 2007 in a garden near Paarl in the Western Cape (WC) Province of South Africa. Another occurrence was found during November 2007 on daylily plants in a garden near Franschhoek (WC). Upon further investigation, diseased daylily plants were found during February 2008 in nurseries in Stellenbosch, Buffeljagsrivier, and George (WC). The cultivars that have been infected include Laura Lane, Anna Mae Hager, and Russian Rhapsody. Symptoms included yellow-to-brown streaks on the leaves. Leaves had small chlorotic spots on the adaxial side, and on the abaxial side, were many small orange-to-yellow erumpent pustules with yellow-to-orange, pulverulent urediniospores. The rust fungus was identified as Puccinia hemerocallidis Thüm. The morphology matches that given in Hernández et al. (1). Urediniospores were globose to ellipsoid with yellow contents and measured 22 to 31 × 16 to 26 µm (mean 25.5 × 22 µm, n = 25). Spore walls were echinulate, hyaline, 2 to 3 µm thick, and with obscure germ pores. No teliospores were observed. Herbarium specimens have been lodged in the South African National Fungus Collection in Pretoria (PREM 59814, 59855, and 59856). The alternative host, Patrinia spp., is not endemic to South Africa and according to several nurseries in the WC, also not sold. P. hemerocallidis has previously been reported from eastern Asia, the United States, and Costa Rica (1,2). To our knowledge, this is the first report of P. hemerocallidis on daylilies in South Africa. References: (1) J. R. Hernandez et al. Plant Dis. 86:1194, 2002. (2) J. L. Williams-Woodward et al. Plant Dis. 85:1121, 2001.

First Report of a Peronospora Species on Sweet Basil in South Africa.

Sweet basil (Ocimum basilicum) is an herbaceous aromatic annual plant of the family Lamiaceae grown for its flavoring and fragrances that can be used fresh or dried. In South Africa, sweet basil is grown on a commercial scale. Downy mildew has recently been reported as one of the most destructive diseases of sweet basil in Switzerland, France, and Italy (1-3). The identity of the downy mildew species infecting sweet basil has been controversial and has been indicated as Peronospora lamii, a presumably undescribed (unnamed) Peronospora species, as well as a few species of which the status as distinct species is mostly unclear or doubtful (1). The distinction between P. lamii and the unnamed Peronospora species has been based on their sporangial dimensions, with P. lamii having sporangial dimensions with a length and width range of 16 to 26 × 15 to 23 µm (average 21 × 18 µm) and the unnamed Peronospora species having sporangial dimensions of 20 to 35 × 15 to 25 µm (average 28 × 22 µm) (1) or 23 to 36 × 18 to 29 µm (average 29 × 23 µm) (2). Additionally, internal transcribed spacer (ITS) sequence data has also been used to show that P. lamii and the unnamed Peronospora species on basil are not similar (1). In the Western Cape Province of South Africa, a sweet basil sample was received at the Stellenbosch University Plant Disease Clinic in 2005 from a grower in the region who experienced almost 50% crop failure under greenhouse-grown conditions. Initial symptoms were chlorotic leaves that subsequently developed a brown sporulation on the abaxial side. Microscopic observations of the brown sporulation were consistent with a Peronospora species. The sporangiophores branched two to five times with lengths ranging from 130 to 290 µm (average 194 µm). Sporangiophores terminated with dichotomously branched denticels bearing single detachable sporangia. Sporangia measured 26 to 34 × 20 to 28 µm (average 30 × 24 µm) and were elliptical and brown. The sporangia were similar in shape, color, and size range as that previously reported for a unnamed Peronospora species on sweet basil (1,2). Sequence analyses were also conducted on two isolates by first extracting DNA from spores that were washed from leaves using the Wizard SV genomic DNA purification system (Promega, Madison, WI), followed by polymerase chain reaction (PCR) amplification and sequencing of the ITS1, 5.8S, and ITS2 regions using primers ITS6 and ITS4 (4). The sequences of the two isolates were identical (GenBank Accession No. DQ479408). BLAST analyses of the sequences revealed a 99% similarity to the unnamed Peronospora species that was isolated from sweet basil in Switzerland and Italy (1). The sequences of the South African isolates only had low homology to P. lamii. To our knowledge, this is the first report of a Peronospora species on sweet basil in South Africa that on the basis of morphology and ITS sequence data is similar to the unnamed Peronospora species recently described in Switzerland and Italy on sweet basil (1). References: (1) L. Belbahri et al. Mycol. Res. 109:1276, 2005. (2) A. Garibaldi et al. Plant Dis. 88:312, 2004. (3) A. Garibaldi et al. Plant Dis. 89:683, 2005. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds., Academic Press, San Diego, 1990.

Effect of phlebotomy on the ferritin iron content in the rat liver as determined morphometrically with the use of electron energy loss spectroscopy.

Phlebotomy of untreated and iron-loaded rats results in a significant decrease in total liver iron. In iron-loaded rats a marked decrease in iron-containing particles is observed ultrastructurally in lysosomes and cytoplasm of hepatic sinusoidal cells but not in parenchymal cells. This remarkable phenomenon was further investigated in a morphometric study, based on element-specific (iron) distribution images made in situ in the parenchymal cell by means of electron energy loss spectroscopy. With the use of this technique it could be shown that in spite of phlebotomy the ferritin iron content of the iron-loaded liver parenchymal cell is not decreased.

Studies on ferritin in rat liver and spleen during repeated phlebotomy.

1. The ferritin content of liver and spleen in normal and iron-loaded rats decreased during repeated phlebotomy. 2. During increased iron demand, ferritin is degraded in toto. 3. With the ESI and EELS technique the iron distribution was followed in different cell types and cellular compartments. 4. We have demonstrated two methods of iron mobilisation: (a) catabolism of lysosomal ferritin in toto and (b) delivery of ferritin from parenchymal cell into the bile and degradation of ferritin in toto.