Elucidating key plant growth-promoting (PGPR) traits in Burkholderia sp. Nafp2/4-1b (=SARCC-3049) using gnotobiotic assays and whole-genome-sequence analysis.

Burkholderia sp. Nafp2/4-1b (=SARCC-3049) is a plant growth-promoting rhizobacteria (PGPR) initially isolated from the rhizosphere of pristine grassland in South Africa, and its ability to enhance growth was previously evaluated on maize (Zea mays L.). Here, the bacterium was tested with the aim of investigating its role in improving the nodulation and growth of the forage legume lucerne (Medicago sativa L.) when it is co-inoculated with the rhizobial symbionts of this legume in the glasshouse. When the co-inoculation resulted in a statistically significant (P = 0·05) increase in the number of nodules and improved plant biomass compared with single inoculation, we sequenced and analysed its genome to gain a better understanding of the genetic determinants responsible for the observed PGPR traits. The Illumina HiSeq 2500-sequenced genome resulted in 92 scaffolds, with an N50 of 322 407 bp, a total draft genome size of 7 788 045 bp and GC content of 66·2%. Analysis of the genome sequence confirmed the presence of a number of essential genes that code for various PGPR traits. The main plant beneficial genes associated with PGPR traits in Burkholderia sp. Nafp2/4-1b include pyoverdine siderophores biosynthesis gene (PvdF); acdS that codes for 1-aminocyclopropane-1-carboxylate (ACC) deaminase; the tryptophan synthase genes involved in auxin biosynthesis (TSA1, TSB1) and the pqqABCDE operon related to phosphate solubilization. This study generated valuable information on the potential of the PGPR Burkholderia sp. strain Nafp2/4-1b as an effective commercial inoculant, which warrants further formulation and field application studies before developing it into a low cost, environmentally safe and effective biofertilizer.

Genera of phytopathogenic fungi: GOPHY 1.

Genera of Phytopathogenic Fungi (GOPHY) is introduced as a new series of publications in order to provide a stable platform for the taxonomy of phytopathogenic fungi. This first paper focuses on 21 genera of phytopathogenic fungi: Bipolaris, Boeremia, Calonectria, Ceratocystis, Cladosporium, Colletotrichum, Coniella, Curvularia, Monilinia, Neofabraea, Neofusicoccum, Pilidium, Pleiochaeta, Plenodomus, Protostegia, Pseudopyricularia, Puccinia, Saccharata, Thyrostroma, Venturia and Wilsonomyces. For each genus, a morphological description and information about its pathology, distribution, hosts and disease symptoms are provided. In addition, this information is linked to primary and secondary DNA barcodes of the presently accepted species, and relevant literature. Moreover, several novelties are introduced, i.e. new genera, species and combinations, and neo-, lecto- and epitypes designated to provide a stable taxonomy. This first paper includes one new genus, 26 new species, ten new combinations, and four typifications of older names.

Finding the missing link: Resolving the Coryneliomycetidae within Eurotiomycetes.

Species belonging to the Coryneliaceae and parasitizing Podocarpaceae hosts were collected from different locations in South Africa and studied morphologically by light microscopy and molecularly by obtaining partial nrDNA (ITS-1/5.8S/ITS-2, 18S and 28S) gene sequences. The position of the Coryneliaceae within the Eurotiomycetidae was not confirmed and a new subclass, Coryneliomycetidae, was introduced. While Eurotiomycetidae usually form cleistothecia/gymnothecia with evanescent, unitunicate asci, and Chaetothyriomycetidae mostly perithecia with bitunicate/fissitunicate to evanescent asci, Coryneliomycetidae form pseudothecial mazaedial ascomata, initially with double-walled asci with the outer layer deliquescing, resulting in passive ascospore release. The Coryneliomycetidae thus occupies a unique position in the Eurotiomycetes. Furthermore, epitypes were designated for Corynelia uberata, the type species of Corynelia (type genus of the family, order and subclass), Lagenulopsis bispora, the type species of Lagenulopsis, and Tripospora tripos the type species of Tripospora, with Lagenulopsis and Tripospora confirmed as belonging to the Coryneliaceae. Corynelia uberata resolved into three clades, one on Afrocarpus (= Podocarpus) falcatus and A. gracilior, and two clades occurring on P. latifolius, herein described as C. africana and C. fructigena. Morphologically these three species are not readily distinguishable, although they differ in spore dimensions, ascomata shape, ornamentation and DNA phylogeny. It is likely that several more species from other parts of the world are currently erroneously placed in C. uberata.

Shot-Hole Reaction of Trichilia emetica in Response to Infection by Cocconia concentrica in South Africa.

Natal mahogany (Trichilia emetica Vahl) is native to South, Central, and East Africa. In South Africa, it is widely planted as an ornamental and street tree; its wood is used for furniture and the seeds used for medicinal oil. In June 2010, during the Southern Hemisphere winter, severe leaf damage resembling insect herbivory was observed on the leaves of a T. emetica specimen in a subtropical garden in White River, Mpumalanga Province, South Africa (25°19'37.98″S, 33°00'19.47″E, elevation 959 m). However, closer inspection revealed that the damage was not insect related but the result of a pronounced shot-hole reaction of the leaves in response to a fungal infection. Concentric patterns on infected leaves facilitated easy macroscopic identification of the pathogen as Cocconia concentrica (Syd.) Syd. Microscopic characteristics of the fungus associated with these patterns also corresponded to descriptions of C. concentrica by Inácio and Minter (1) and Theissen and Sydow (3): asci were cylindrical-clavate, mostly eight spored, and 62 to 65 × 12 to 15 µm; ascospores were light brown, elliptic to oblong, mostly one-septate, usually wider at one end, somewhat constricted at septum, and 16 to 18.5 × 6 to 7.5 µm; paraphyses were longer than asci, septate, 2.5 µm in diameter, and hyaline but brownish at the rounded, often swollen tips. The occurrence of C. concentrica on Trichilia spp. and other hosts has been well documented, but only as a leaf-spot parasite without any reference to the prominent shot-hole reaction, which to our knowledge, is reported here for the first time. After infection, which is presumably by airborne ascospores (1), scattered or coalescing, relatively round black spots, up to 7.5 mm in diameter, developed on the adaxial leaf surface. Each spot is composed of many circular to elliptical, black, coalescing stromatic ascomata conspicuously arranged in centrifugally expanding concentric rings, later appearing to be covered by an ascoma-bearing crust. Eventually, the formation of an abscission layer, generally along the outer edge of the outer ring or some distance away, may be triggered in the leaf tissue along the periphery of each spot, separating and isolating the infected leaf area from the rest of the lamina. Infected areas eventually slough off from the rest of the leaf (but may remain attached more intimately and longer along veins) and fall out, leaving gaping circular to irregular shot-holes edged by a prominent corky rim. A similar phenomenon has been reported and illustrated for T. connaroides infected by Phloeospora trichiliae in India (2) and T. tuberculata infected by an unidentified ascomycete in Panama ( http://researchwatch.net/nsf_grants/9902346 ). Shot-hole formation as a self-defense mechanism against fungal infection thus appears to be a widely occurring generic character in Trichilia, and it is remarkable that this symptom, subsequently witnessed elsewhere in Mpumalanga, has apparently gone unnoticed in Africa until now and that it has received no mention in the abundant literature on the trees of the region. Voucher material (PREM 60718) has been deposited at the National Collection of Fungi, Pretoria, South Africa. References: (1) C. A. Inácio and D. W. Minter. IMI Descriptions of Fungi and Bacteria. 145 (1445), 2002. (2) M. D. Mehrotra and R. K. Verma. Mycol. Res. 98:1192, 1994. (3) F. Theissen and H. Sydow. Ann. Mycol. 13:149, 1915.

Claviceps cyperi, a new cause of severe ergotism in dairy cattle consuming maize silage and teff hay contaminated with ergotised Cyperus esculentus (nut sedge) on the Highveld of South Africa.

During December/January 1996/97 typical summer syndrome (hyperthermia and a 30% drop in milk yield) occurred in succession in two Holstein dairy herds (n=240 and n=150 milking cows, respectively) on the South African Highveld. These farms are situated in the midst of the prime maize and dairy farming areas of South Africa where this condition had never been diagnosed before. The individual components of the concentrate on both farms were negative for ergot alkaloids. Endophytic fungi and/or ergot infestation of teff and other grasses fed to the cows were then suspected of being involved, but neither endophytes nor ergot alkaloids could be implicated from these sources. By measuring the serum prolactin levels of groups of sheep (n=5) fed the first farm's total mixed ration (TMR) or its three individual fibre components for a period of 11 days, the source of the ergot alkaloids was identified. A statistically significant decrease in the level of this hormone occurred only in the group on maize silage (which constituted 28% on dry matter base of the TMR). The involvement of the maize silage was further chemically confirmed by the high levels of total ergot alkaloids, predominantly ergocryptine, found by LC-MS in the silage as well as in the TMR (115-975 ppb and 65-300 ppb, respectively). The ergot alkaloid content (mainly ergocryptine) of the maize silage on the second affected farm was 875 ppb. Withdrawal of contaminated silage resulted in gradual recovery of stock on both farms. Nut sedge (Cyperus esculentus and Cyperus rotundus of the family Cyperaceae) has a world-wide distribution and is a common weed in annual crops, and can be parasitized by Claviceps cyperi. Careful examination of the maize silage from both farms revealed that it was heavily contaminated with nut sedge and that it contained minute sclerotia, identified as those of Claviceps cyperi, originating from the latter. Nut sedge was abundant on both farms and it is believed that late seasonal rain had resulted in mature, heavily ergotised nut sedge being cut with the silage. Claviceps cyperi sclerotia, collected on the affected fields in the following autumn contained 3600-4000 ppm ergocryptine. That the dominant alkaloid produced by this particular fungus was indeed ergocryptine, was confirmed by negative ion chemical ionization MS/MS. In one further outbreak in another Holstein herd, teff hay contaminated with ergotised nut sedge and containing 1200 ppb alkaloids, was incriminated as the cause of the condition. This is the first report of bovine ergotism not associated with the Poaceae infected with Claviceps purpureum or endophytes but with the family Cyperaceae and this particular fungal phytopathogen.

First Report of Ascochyta spp. on Soybean in South Africa.

During February 2000 soybean fields over a wide area in South Africa were affected by a previously unreported disease. A typical target spot developed on leaves and many became blighted. Elongated dark brown to black lesions developed on the stems, often resulting in wilting of young shoots. Petioles and leaf axils were colonized, resulting in premature leaf drop. The most severe manifestation was on pedicels where infection suppressed podfill and plants remained green as ripening was delayed. Two distinct groups of isolates were obtained from lesions. In both groups an Ascochyta anamorph was present, while from some lesions collected in Kwazulu-Natal and Mpumalanga, a teliomorph (Mycosphaerella) was also present. Since dry beans are produced in these areas and M. phaseolorum is common on this crop, it was believed that the pathogen might have moved to soybean. In cross inoculations, both soybean and dry bean isolates were pathogenic to both hosts. However, M. phaseolorum isolates were more aggressive to both hosts than the Ascochyta sp. Morphologically the anamorphs of two types of isolates were indistinguishable. DNA was isolated from freeze dried mycelia using a modified version of the CTAB-method described by Graham et al. (1). The DNA concentration and purity were estimated by measuring absorbances at A260 and A280. Genetic difference between both isolates were determined using amplified fragment length polymorphism (AFLP) technique. The AFLP analysis was performed following the protocol described by Vos et al. (2) and the product manual supplied by Life Technologies Inc. (Gaithersburg, MD) with minor modifications. Five randomly selected primer pair combinations were tested for their ability to reveal polymorphisms between the isolates. The gel electrophoresis for AFLP products was as described by Vos et al. (2). AFLP gels were silver stained following the protocol described by silver sequence DNA sequencing system manual (Promega, Madison, WI). All five primer pairs revealed only polymorphisms between isolates. No corresponding bands between the two isolates were detected using these five primer pair combinations. It is concluded that both M. phaseolorum and an unidentified Ascochyta sp. were the cause of the epidemic. Ascochyta spp. have not previously been reported on soybean in South Africa. References: (1) G. C. Graham et al. Biotechniques 16:48-50, 1994. (2) P. Vos et al. Nucleic Acids Res. 21:4407-4414, 1995.