First Report of Puccinia triticina (Leaf Rust) Race FBPT on Wheat in South Africa.

Eleven isolates of Puccinia triticina Erikss. collected from bread wheat (Triticum aestivum L.) in the Western Cape during the 2010 annual rust survey were pathotyped to a race not previously recorded in South Africa. Replicated race analysis on seedlings of 16 Thatcher (Tc) near-isogenic differential lines at two rust laboratories confirmed avirulence (infection types [ITs] 0; to 2+) for lines with Lr1, Lr2a, Lr9, Lr11, Lr16, Lr24, and Lr26 and virulence (ITs 3 to 4) for lines with Lr2c, Lr3, Lr3ka, Lr10, Lr14a, Lr17, Lr18, Lr30, and LrB. Thatcher lines with LrB, Lr10, Lr14a, and Lr18 were added as the fourth set to the 12 original differential lines (1,2). This profile codes to race FBPT according to the North American system, and, based on these differentials, resembles a P. triticina isolate from Gwebi, Zimbabwe, in 2012 (Z. A. Pretorius, unpublished data). When additional single gene lines were tested with FBPT (race 3SA147 according to the ARC-Small Grain Institute rust notation procedure), lines with Lr2b, Lr15, Lr19, Lr20, Lr21, Lr25, Lr27+31, Lr28, Lr29, Lr32, Lr36, Lr38, Lr45, Lr47, Lr50, Lr51, and Lr52 were effective, whereas lines with Lr3bg, Lr23, Lr28, and Lr33 were ineffective. In adult plant tests in a greenhouse, Thatcher lines containing Lr12 (IT ;1c), Lr13 (IT ;1c), Lr22a (IT 1), Lr35 (IT ;1+), and Lr37 (IT ;1) were resistant, whereas Thatcher (Lr22b) was susceptible. Of 146 South African cultivars and lines infected as seedlings with 3SA147 (FBPT), 83% were resistant (IT ≤ 2) and 5% showed within-line variation. Entries showing compatible ITs with 3SA147 (FBPT) were also susceptible to either or both of 3SA133 (PDRS) and 3SA146 (MCDS). In addition, the new race was genotyped using 16 simple sequence repeat (SSR) primer-combinations (3). Three single pustule isolates of 3SA147 (FBPT) were identical and showed 82% and 76% similarity with the recently described races 3SA146 (MCDS) and 3SA145 (CCPS), respectively (4). Minimum spanning network analysis confirmed this close genetic relationship among the three races. However, since their virulence phenotypes differ, it is proposed that 3SA147 (FBPT) is not a stepwise mutation from either 3SA145 (CCPS) or 3SA146 (MCDS), but rather a foreign introduction into South Africa. As most current breeding lines and wheat cultivars are resistant, it is unlikely that race FBPT will threaten wheat production in South Africa, but its detection underlines the fact that new P. triticina variants have been occurring at regular intervals in the region. References: (1) J. A. Kolmer et al. Austr. J. Agric. Res. 58:631, 2007. (2) D. L. Long and J. A. Kolmer. Plant Dis. 79:525, 1989. (3) L. J. Szabo and J. A. Kolmer. Mol. Ecol. Notes 7:708, 2007. (4) T. Terefe et al. Plant Dis. 95:611, 2011.

First Report of a New TTKSF Race of Wheat Stem Rust (Puccinia graminis f. sp. tritici) in South Africa and Zimbabwe.

Seven races have been described in the Ug99 race group of Puccinia graminis f. sp. tritici (2). Ug99-related races previously recorded in South Africa are TTKSF, TTKSP, and PTKST (4). In December 2010, severe stem rust infection of the winter wheat cv. Matlabas was observed for the first time in South Africa. Race analysis using the 20 North American (NA) stem rust differential lines and letter code system classified the race as TTKSF. In comparative infection studies in a greenhouse, cv. Matlabas seedlings were susceptible (infection type [IT] 4) to isolate UVPgt61/1 (TTKSF+) collected from Afrikaskop in the eastern Free State, whereas the cultivar was resistant (IT 1 to 2) to stem rust isolates 2013 (TTKSF), UVPgt55 (TTKSF), UVPgt59 (TTKSP), and UVPgt60 (PTKST). Isolate 2013 represents the original collection of race TTKSF in South Africa (1). In addition to the NA differentials, no variation in the IT range of seedlings of lines with Sr7a, 8b, 12, 13, 14, 16, 18, 19, 22, 25, 26, 27, 28, 29, 32, 33, 34, 35, 39, 41, 42, 43, 44, Em, R, Tt2, and Satu was observed between UVPgt61/1 and UVPgt55. With the exception of cv. Matlabas, ITs of 106 South African cultivars likewise did not differentiate UVPgt61/1 and UVPgt55. Seedling IT studies were conducted at least twice. Microsatellite analysis (4) showed that all single pustule isolates established from the original Matlabas isolate formed part of the Ug99 group. When characterized with selected single nucleotide polymorphisms (SNPs), all single pustule isolates shared an identical genotype that differed from UVPgt55 (TTKSF), a foreign introduction into South Africa (1,3). SNP genotype analysis suggests that UVPgt61/1 is genetically dissimilar to UVPgt55, as is Zim1009, another TTKSF+ isolate that was collected from Birchenough in Zimbabwe. Studies are underway to determine the identity of the defeated Sr gene in Matlabas and the cultivar has been added to the South African stem rust differential set. TTKSF+ is the eighth race detected in the Ug99 group. Since no other cultivars or advanced lines were found to carry the Matlabas gene, it is unlikely that race TTKSF+ will threaten wheat production in South Africa. However, the occurrence of a new Ug99-related race emphasizes the variability within this internationally important group. References: (1) W. H. P. Boshoff et al. Plant Dis. 86:922, 2002. (2) R. F. Park et al. Euphytica 179:109, 2011. (3) B. Visser et al. Mol. Plant Pathol. 10:213, 2009. (4) B. Visser et al. Euphytica 179:119, 2011.

First Report of a New Wheat Leaf Rust (Puccinia triticina) Race with Virulence for Lr12, 13, and 37 in South Africa.

A new race of Puccinia triticina was collected from common wheat (Triticum aestivum) in the Eastern and Western Cape provinces during the annual rust survey in 2009. Six single-pustule isolates from a field collection, which were shown to be a new race in preliminary analyses, were inoculated onto seedlings of 16 Thatcher (Tc) near-isogenic differential lines (1) and other tester lines with known Lr genes. Standard procedures for inoculation, incubation, and rust evaluation were followed (4) and all infection studies were repeated. The low infection type of Lr18 was confirmed at 18°C. All six isolates were avirulent (infection types [ITs] 0; to 2) to Lr1, 2a, 2c, 9, 11, 16, 18, and 24 and virulent (ITs 3 to 4) to Lr3, 3ka, 10, 14a, 17, 26, 30, B, and Tc (control). The new race, named 3SA145 according to the ARC-Small Grain Institute notation, corresponds to race CCPS in the North American system (1). On the basis of seedling ITs of the extended Lr gene set, 3SA145 was avirulent (ITs 0; to 22+) to Lr2b, 19, 21, 23, 25, 28, 29, 32, 36 (E84081), 38, 45, 47 (KS90H450), 50 (KS96WGRC36), 51 (R05), and 52 and virulent to Lr3bg, 15, 20 (Thew), 27+31 (Gatcher), and 33. Lines containing the adult plant resistance (APR) genes Lr12 (RL6011, IT 3++), Lr13 (CT263, IT 3), Lr22b (Tc, IT 4), and Lr37 (RL6081, IT 3) were susceptible in the adult stage to 3SA145, whereas lines with the APR genes Lr22a (RL6044, IT ;1), Lr34 (RL6058, IT Z1), and Lr35 (RL6082, IT ;1) were resistant in controlled infection studies in a greenhouse. A control, the common race (3SA133), was virulent only on Tc adult plants. In seedlings, 3SA133 was avirulent to Lr15, 17, 26, and 27+31, but unlike 3SA145, it was virulent to Lr1, 2c, 11, 18, 24, and 28. Races 3SA133 and 3SA145 did not differ in their virulence to the remaining seedling genes. Virulence to Lr37 has been reported in several countries, including Australia, Canada, Uruguay, and the United States (1,2). Prior to the detection of 3SA145, adult plants of RL6081 were resistant to all wheat leaf rust races in South Africa. In 2009, however, RL6081 showed severity levels of up to 30S at certain Western Cape trap plot sites. Of 124 South African bread wheat cultivars and advanced breeding lines tested at the seedling stage, 3SA145 was virulent to 48, whereas 3SA133 was virulent to 36 entries. A further six entries were heterogeneous in their reaction to 3SA145. In adult plant infection studies of 48 South African spring wheats in a greenhouse, 19 were susceptible (flag leaf IT ≥3) and 22 were resistant to 3SA145. Seven entries showed a Z3 flag leaf IT indicating adult plant resistance. According to a simple sequence repeat (SSR) study using 17 primer-pair combinations described by Szabo and Kolmer (3), 3SA145 showed 30% homology with the dominant South African races. Although virulence to Lr12 and Lr13 has been known in different leaf rust races in South Africa, to our knowledge, this is the first report of combined virulence to Lr12, 13, and 37. The SSR data and unique avirulence/virulence profile suggest that 3SA145 may be an exotic introduction to South Africa. References: (1) J. A. Kolmer et al. Plant Dis. 89:1201, 2005. (2) B. McCallum and P. Seto-Goh. Can. J. Plant Pathol. 31:80, 2009. (3) L. Szabo and J. Kolmer. Mol. Ecol. Notes 7:708, 2007. (4) T. Terefe et al. S. Afr. J. Plant Soil 26:51, 2009.

Detection of Variants of Wheat Stem Rust Race Ug99 (Puccinia graminis f. sp. tritici) in Zimbabwe and Mozambique.

The migration of Ug99 variants of Puccinia graminis f. sp. tritici is of concern to global wheat production (1). Seven races have been characterized in the Ug99 lineage (3), three of which occur in South Africa (4). During surveys of wheat fields for Ug99 in Zimbabwe and Mozambique in August and September 2010, high stem rust severities were found at Chiredzi, Chisumbanje, and Birchenough in Zimbabwe and at Rotanda in Mozambique. Stem rust was widespread in the lowlands (<800 m above sea level) of Zimbabwe and trace amounts were present in the mid-altitude areas. In Mozambique, stem rust was only observed at Rotanda (sample Moz1001). Collections from Chiredzi (samples Zim1004 and Zim1005), Chisumbanje (Zim1006), and Birchenough (Zim1009 and Zim1010) yielded viable urediniospores for infection studies. According to race analysis conducted on seedlings of the North American stem rust differential set (2) in a greenhouse at 18 to 25°C, Zim1005 and Zim1006 were typed as PTKST and Zim1004 and Zim1009 as TTKSF. Both TTKSF and PTKST were detected in the Zim1010 sample. Race analysis experiments were conducted three times. Urediniospores of isolate Moz1001 were not viable in infection studies, but yielded fungal DNA for simple sequence repeat (SSR) analysis. Using eight selected SSR primer combinations (4), all six isolates clustered within the Ug99 lineage. Isolates Zim1005, Zim1006, Zim1009, Zim1010, and Moz1001 and the stem rust control races TTKSF, TTKSK, and PTKST grouped into two main clusters, with Zim1009 and Zim1010 clustering together and sharing 88% similarity with the rest of the isolates. Zim1005 and Zim1006 were identical to TTKSF and TTKSK, respectively. Zim1004 shared 96% genetic similarity with the TTKSP control, with these two sharing 74% genetic similarity with the remaining isolates. The SSR data correlated with the infection data, except for Zim1004, which was typed as TTKSF but clustered close to TTKSP. Wheat cvs. SC Nduna, SC Shine, SC Stallion, SC Smart, Kana, Insiza, and Dande are predominant in Zimbabwe. Cv. SC Stallion and other unidentified cultivars were susceptible to P. graminis f. sp. tritici in the field in Zimbabwe. In Mozambique, the tall, local cv. Sitsonko was susceptible to P. graminis f. sp. tritici but no infections were observed on SC Nduna or SC Shine. The similarity in P. graminis f. sp. tritici races in Zimbabwe, South Africa, and Mozambique suggests that inoculum is exchanged within the region and explains the detection of race PTKST in South Africa in 2009. Trajectory models showed winds originating at Birchenough in October 2009, where stem rust was observed, passing directly over KwaZulu-Natal, South Africa within 48 to 72 h. Race PTKST was confirmed from collections in KwaZulu-Natal in November 2009 (4). The confirmation of Sr31 virulence in race PTKST in Zimbabwe is important because it provides new geographical records for an Ug99-related race and puts Southern African cultivars with 1B.1R resistance at risk. References: (1) D. Hodson. Euphytica 179:93, 2011. (2) Y. Jin et al. Plant Dis. 92:923, 2008. (3) R. F. Park et al. Euphytica 179:109, 2011. (4) B. Visser et al. Euphytica 179:119, 2011.

Genetic structure and relationships within and between cultivated and wild sorghum (Sorghum bicolor (L.) Moench) in Kenya as revealed by microsatellite markers.

Understanding the extent and partitioning of diversity within and among crop landraces and their wild/weedy relatives constitutes the first step in conserving and unlocking their genetic potential. This study aimed to characterize the genetic structure and relationships within and between cultivated and wild sorghum at country scale in Kenya, and to elucidate some of the underlying evolutionary mechanisms. We analyzed at total of 439 individuals comprising 329 cultivated and 110 wild sorghums using 24 microsatellite markers. We observed a total of 295 alleles across all loci and individuals, with 257 different alleles being detected in the cultivated sorghum gene pool and 238 alleles in the wild sorghum gene pool. We found that the wild sorghum gene pool harbored significantly more genetic diversity than its domesticated counterpart, a reflection that domestication of sorghum was accompanied by a genetic bottleneck. Overall, our study found close genetic proximity between cultivated sorghum and its wild progenitor, with the extent of crop-wild divergence varying among cultivation regions. The observed genetic proximity may have arisen primarily due to historical and/or contemporary gene flow between the two congeners, with differences in farmers' practices explaining inter-regional gene flow differences. This suggests that deployment of transgenic sorghum in Kenya may lead to escape of transgenes into wild-weedy sorghum relatives. In both cultivated and wild sorghum, genetic diversity was found to be structured more along geographical level than agro-climatic level. This indicated that gene flow and genetic drift contributed to shaping the contemporary genetic structure in the two congeners. Spatial autocorrelation analysis revealed a strong spatial genetic structure in both cultivated and wild sorghums at the country scale, which could be explained by medium- to long-distance seed movement.

Identification and mapping of AFLP markers linked to peanut (Arachis hypogaea L.) resistance to the aphid vector of groundnut rosette disease.

Groundnut rosette disease is the most destructive viral disease of peanut in Africa and can cause serious yield losses under favourable conditions. The development of disease-resistant cultivars is the most effective control strategy. Resistance to the aphid vector, Aphis craccivora, was identified in the breeding line ICG 12991 and is controlled by a single recessive gene. Bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) analysis were employed to identify DNA markers linked to aphid resistance and for the development of a partial genetic linkage map. A F(2:3) population was developed from a cross using the aphid-resistant parent ICG 12991. Genotyping was carried out in the F2 generation and phenotyping in the F3 generation. Results were used to assign individual F2 lines as homozygous-resistant, homozygous-susceptible or segregating. A total of 308 AFLP (20 EcoRI+3/MseI+3, 144 MluI+3/MseI+3 and 144 PstI+3/MseI+3) primer combinations were used to identify markers associated with aphid resistance in the F(2:3) population. Twenty putative markers were identified, of which 12 mapped to five linkage groups covering a map distance of 139.4 cM. A single recessive gene was mapped on linkage group 1, 3.9 cM from a marker originating from the susceptible parent, that explained 76.1% of the phenotypic variation for aphid resistance. This study represents the first report on the identification of molecular markers closely linked to aphid resistance to groundnut rosette disease and the construction of the first partial genetic linkage map for cultivated peanut.

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.

Genetic Variability Within and Among Mycelial Compatibility Groups of Sclerotium rolfsii in South Africa.

ABSTRACT Isolates of Sclerotium rolfsii, the causal organism of stem rot or southern blight of groundnut, can be placed in mycelial compatibility groups (MCGs) based on hyphal interactions between isolates. The aim of this study was to determine whether amplified fragment length polymorphism (AFLP) analysis was a suitable technique to assess genetic variability between isolates and MCGs of S. rolfsii. For preliminary genetic analysis, 10 isolates were selected from each of two MCGs and compared with each other using the restriction enzymes EcoRI and MseI and 4 primer pairs. The number of polymorphisms ranged from 10 to 36 per primer combination, with an average of 22.5. AFLP analysis clearly showed genotypic differences (22%) among MCGs B and C, with a maximum variation of 6.41% between any two isolates per group using four primer pairs. Certain isolates could not be distinguished from each other. A more in-depth study of 10 isolates from MCG B, using 8 additional primer pairs, showed small genetic differences (maximum of 4.2% and minimum of 0.2%) between isolates. These results suggested that DNA could be pooled for comparison of MCGs. Pooled DNA from isolates within groups using 20 primer pairs confirmed differences between 9 MCGs. This technique effectively differentiated MCGs of S. rolfsii from each other and also detected differences between isolates within a single MCG.