Nicolaas Albertus Van der Merwe

IMA Genome-F 4: Draft genome sequences of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygamai, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata.

The genomes of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygami, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata are presented in this genome announcement. These seven genomes are from endophytes, plant pathogens and economically important fungal species. The genome sizes range from 26.6 Mb in the case of Leptographium lundbergii to 44 Mb for Chrysoporthe austroafricana. The availability of these genome data will provide opportunities to resolve longstanding questions regarding the taxonomy of species in these genera, and may contribute to our understanding of the lifestyles through comparative studies with closely related organisms.

Evolution of the tissue factor pathway inhibitor-like Kunitz domain-containing protein family in Rhipicephalus microplus

One of the principle mechanisms utilised by ticks to obtain a blood meal is the subversion of the hosts haemostatic response. This is achieved through the secretion of saliva containing anti-haemostatic proteins into the feeding lesion. Lineage-specific expansion of predicted secretory protein families have been observed in all previously studied ticks and occurred in response to adaptation to a blood-feeding environment. Of these, the predominant families are common between both hard and soft ticks. One of these families, namely the Kunitz domain-containing protein family, includes proven tissue factor pathway inhibitor-like (TFPI-like) anti-haemostatics such as ixolaris and penthalaris that play a crucial role during tick feeding. Although Kunitz-type proteins have been found in Rhipicephalus microplus, the TFPI-like Kunitz protein family has not yet been studied. We report a comprehensive search for TFPI-like Kunitz domain-containing proteins in R. microplus expressed sequence tag libraries, resulting in the identification of 42 homologues. The homologues were bioinformatically and phylogenetically studied, including the application of an intensive Bayesian Markov Chain Monte Carlo (MCMC) analysis of the individual Kunitz domain nucleotide sequences. We show that the R. microplus TFPI-like Kunitz protein family groups into two main clades that presumably underwent ancient duplication, which indicates that a whole genome duplication event occurred at least 150 million years ago. Evidence for recent and ancient gene and domain duplication events was also found. Furthermore, the divergence times of the various tick lineages estimated in this paper correspond with those presented in previous studies. The elucidation of this large protein familys evolution within R. microplus adds to current knowledge of this economically important tick.

Evidence for inter-specific recombination among the mitochondrial genomes of Fusarium species in the Gibberella fujikuroi complex

BackgroundThe availability of mitochondrial genomes has allowed for the resolution of numerous questions regarding the evolutionary history of fungi and other eukaryotes. In the Gibberella fujikuroi species complex, the exact relationships among the so-called “African”, “Asian” and “American” Clades remain largely unresolved, irrespective of the markers employed. In this study, we considered the feasibility of using mitochondrial genes to infer the phylogenetic relationships among Fusarium species in this complex. The mitochondrial genomes of representatives of the three Clades (Fusarium circinatum, F. verticillioides and F. fujikuroi) were characterized and we determined whether or not the mitochondrial genomes of these fungi have value in resolving the higher level evolutionary relationships in the complex.ResultsOverall, the mitochondrial genomes of the three species displayed a high degree of synteny, with all the genes (protein coding genes, unique ORFs, ribosomal RNA and tRNA genes) in identical order and orientation, as well as introns that share similar positions within genes. The intergenic regions and introns generally contributed significantly to the size differences and diversity observed among these genomes. Phylogenetic analysis of the concatenated protein-coding dataset separated members of the Gibberella fujikuroi complex from other Fusarium species and suggested that F. fujikuroi (“Asian” Clade) is basal in the complex. However, individual mitochondrial gene trees were largely incongruent with one another and with the concatenated gene tree, because six distinct phylogenetic trees were recovered from the various single gene datasets.ConclusionThe mitochondrial genomes of Fusarium species in the Gibberella fujikuroi complex are remarkably similar to those of the previously characterized Fusarium species and Sordariomycetes. Despite apparently representing a single replicative unit, all of the genes encoded on the mitochondrial genomes of these fungi do not share the same evolutionary history. This incongruence could be due to biased selection on some genes or recombination among mitochondrial genomes. The results thus suggest that the use of individual mitochondrial genes for phylogenetic inference could mask the true relationships between species in this complex.

Genetic variation among Sclerotium isolates from Benin and South Africa, determined using mycelial compatibility and ITS rDNA sequence data

Damping-off and stem rot of cowpea caused bySclerotium rolfsii has previously been reported in Benin, where the pathogen showed variation in growth and sclerotia production among isolates. Pathogenicity, mycelial compatibility group (MCG) tests and rDNA sequence analyses were conducted on different isolates ofS. rolfsii and S. delphinii collected from different hosts and geographical areas in Benin and South Africa. All the isolates, when inoculated into soil and planted with cowpea, caused damping-off and stem rot symptoms. Aggressiveness among isolates varied depending on the host from which each was isolated. Isolates originating from cowpea produced the highest disease incidence followed by isolates from peanuts. Four MCGs were distinguished among 66 isolates. Isolates from the same hosts tended to group into the same MCG. The incidence of damping-off and stem rot of cowpea, expressed as percentage diseased plants, varied among MCGs. Plants inoculated with MCG2 displayed the highest disease incidence, whereas MCG4 resulted in the least. Parsimony analysis of ITS DNA sequence data supported a close affinity of the Sclerotium spp. but showed genetic variation among isolates with no grouping based on host of origin.

SNP Analysis Infers that Recombination Is Involved in the Evolution of Amitraz Resistance in Rhipicephalus microplus

Rhipicephalus microplus, better known as the Asiatic cattle tick, is a largely invasive ectoparasite of great economic importance due to the negative effect it has on agricultural livestock on a global scale, particularly cattle. Tick-borne diseases (babesiosis and anaplasmosis) transmitted by R. microplus are alarming as they decrease the quality of livestock health and production. In sub-Saharan Africa, cattle represent a major source of meat and milk, but this region of the world is severely affected by the Rhipicephalus microplus tick. The principal method for tick control is the use of chemical acaricides, notably amitraz, which was implemented in the 1990’s after resistance to other acaricides surfaced. However, the efficiency of chemical control is hindered by an increase in the frequency of mutant resistance alleles to amitraz in tick populations. Presently, the only way to assess amitraz resistance is by means of larval packet tests, but this technique is time-consuming and not particularly cost effective. The main aims of this study were three-fold. First, we attempted to correlate two known SNPs in the octopamine/tyramine (OCT/Tyr) receptor with amitraz resistance in South African field samples of R. microplus. Second, we calculated gametic disequilibrium for these SNPs to determine whether they are randomly associated. Lastly, we conducted a study to assess the evolutionary effects of recombination within the OCT/Tyr receptor. Our results confirmed that the two SNPs are associated with amitraz resistance in the South African tick strain, and that they are in gametic disequilibrium. Additionally, recombination was detected in the OCT/Tyr receptor generating two recombinant haplotypes. These results are of concern to farmers in sub-Saharan Africa, and the emergence of amitraz resistance should be closely monitored in future. Therefore, we present a quick and affordable RFLP based diagnostic technique to assess amitraz resistance in field samples of R. microplus.

Genetic diversity, acaricide resistance status and evolutionary potential of a Rhipicephalus microplus population from a disease-controlled cattle farming area in South Africa

The Southern cattle tick, Rhipicephalus microplus is a hematophagous ectoparasite of great veterinary and economic importance. Along with its adaptability, reproductive success and vectoring capacity, R. microplus has been reported to develop resistance to the major chemical classes of acaricides currently in use. In South Africa, the Mnisi community in the Mpumalanga region offers a unique opportunity to study the adaptive potential of R. microplus. The aims of this study therefore included characterising acaricide resistance and determining the level and pattern of genetic diversity for R. microplus in this region from one primary population consisting of 12 communal dip-stations. The level of acaricide resistance was evaluated using single nucleotide polymorphisms (SNPs) in genes that contribute to acaricide insensitivity. Additionally, the ribosomal internal transcribed spacer 2 (ITS2) gene fragments of collected individuals were sequenced and a haplotype network was constructed. A high prevalence of alleles attributed to resistance against formamidines (amitraz) in the octopamine/tyramine (OCT/Tyr) receptor (frequency of 0.55) and pyrethroids in the carboxylesterase (frequency of 0.81) genes were observed. Overall, the sampled tick population was homozygous resistant to pyrethroid-based acaricides in the voltage-gated sodium channel (VGS) gene. A total of 11 haplotypes were identified in the Mnisi R. microplus population from ITS2 analysis with no clear population structure. From these allele frequencies it appears that formamidine resistance in the Mnisi community is on the rise, as the R. microplus populations is acquiring or generating these resistance alleles. Apart from rearing multi-resistant ticks to commonly used acaricides in this community these ticks may pose future problems to its surrounding areas.

Intron Derived Size Polymorphism in the Mitochondrial Genomes of Closely Related Chrysoporthe Species

In this study, the complete mitochondrial (mt) genomes of Chrysoporthe austroafricana (190,834 bp), C. cubensis (89,084 bp) and C. deuterocubensis (124,412 bp) were determined. Additionally, the mitochondrial genome of another member of the Cryphonectriaceae, namely Cryphonectria parasitica (158,902 bp), was retrieved and annotated for comparative purposes. These genomes showed high levels of synteny, especially in regions including genes involved in oxidative phosphorylation and electron transfer, unique open reading frames (uORFs), ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs), as well as intron positions. Comparative analyses revealed signatures of duplication events, intron number and length variation, and varying intronic ORFs which highlighted the genetic diversity of mt genomes among the Cryphonectriaceae. These mt genomes showed remarkable size polymorphism. The size polymorphism in the mt genomes of these closely related Chrysoporthe species was attributed to the varying number and length of introns, coding sequences and to a lesser extent, intergenic sequences. Compared to publicly available fungal mt genomes, the C. austroafricana mt genome is the second largest in the Ascomycetes thus far.

Genome-Wide Macrosynteny among Fusarium Species in the Gibberella fujikuroi Complex Revealed by Amplified Fragment Length Polymorphisms

The Gibberella fujikuroi complex includes many Fusarium species that cause significant losses in yield and quality of agricultural and forestry crops. Due to their economic importance, whole-genome sequence information has rapidly become available for species including Fusarium circinatum, Fusarium fujikuroi and Fusarium verticillioides, each of which represent one of the three main clades known in this complex. However, no previous studies have explored the genomic commonalities and differences among these fungi. In this study, a previously completed genetic linkage map for an interspecific cross between Fusarium temperatum and F. circinatum, together with genomic sequence data, was utilized to consider the level of synteny between the three Fusarium genomes. Regions that are homologous amongst the Fusarium genomes examined were identified using in silico and pyrosequenced amplified fragment length polymorphism (AFLP) fragment analyses. Homology was determined using BLAST analysis of the sequences, with 777 homologous regions aligned to F. fujikuroi and F. verticillioides. This also made it possible to assign the linkage groups from the interspecific cross to their corresponding chromosomes in F. verticillioides and F. fujikuroi, as well as to assign two previously unmapped supercontigs of F. verticillioides to probable chromosomal locations. We further found evidence of a reciprocal translocation between the distal ends of chromosome 8 and 11, which apparently originated before the divergence of F. circinatum and F. temperatum. Overall, a remarkable level of macrosynteny was observed among the three Fusarium genomes, when comparing AFLP fragments. This study not only demonstrates how in silico AFLPs can aid in the integration of a genetic linkage map to the physical genome, but it also highlights the benefits of using this tool to study genomic synteny and architecture.

Genetic analysis of growth, morphology and pathogenicity in the F1 progeny of an interspecific cross between Fusarium circinatum and Fusarium subglutinans

Fusarium circinatum and Fusarium subglutinans are two distinct species in the Gibberella fujikuroi species complex. A genetic linkage map produced from an interspecific cross between these species was used to identify quantitative trait loci (QTLs) associated with variation in mycelial growth and morphology of colony margins (CMs) in the 94 F(1) progeny. Mycelial growth was assessed by measuring culture size at 25°C and 30°C, while CM morphology was characterized in the parents and assessed in their F(1) progeny. In order to test the pathogenicity of the progeny, Pinus patula seedlings were inoculated and lesion lengths were measured after 3weeks. Seven putative QTLs were associated with mycelial growth, three for growth at 25°C and four at 30°C. One highly significant QTL (P<0.001) was present at both growth temperatures. For CM morphology, a QTL was identified at the same position (P<0.001) as the QTL responsible for growth at the two temperatures. The putative QTLs accounted for 45 and 41% of the total mycelial growth variation at 25°C and 30°C, respectively, and for 21% of the variation in CM morphology. Only one of the 94 F(1) progeny was pathogenic on P. patula seedlings. This observation could be explained by the genetic constitution of this F(1) isolate, namely that ∼96% of its genome originated from the F. circinatum parent. This F(1) individual also grew significantly faster at 25°C than the F. circinatum parent (P<0.05), as well as more rapidly than the average growth for the remaining 93 F(1) progeny (P<0.05). However, no association was found between mycelial growth and pathogenicity at 25°C. The highly significant QTL associated with growth at two temperatures, suggests that this is a principal genomic region involved in mycelial growth at both temperatures, and that the same region is also responsible for CM morphology.

Localization and transcriptional responses of Chrysoporthe austroafricana in Eucalyptus grandis identify putative pathogenicity factors

Chrysoporthe austroafricana is a fungal pathogen that causes the development of stem cankers on susceptible Eucalyptus grandis trees. Clones of E. grandis that are partially resistant and highly susceptible have been identified based on the extent of lesion formation on the stem upon inoculation with C. austroafricana. These interactions have been used as a model pathosystem to enhance our understanding of interactions between pathogenic fungi and woody hosts, which may be different to herbaceous hosts. In previous research, transcriptomics of host responses in these two clones to C. austroafricana suggested roles for salicylic acid and gibberellic acid phytohormone signaling in defense. However, it is unclear how the pathogen infiltrates host tissue and which pathogenicity factors facilitate its spread in the two host genotypes. The aim of this study was to investigate these two aspects of the E. grandis–C. austroafricana interaction and to test the hypothesis that the pathogen possesses mechanisms to modulate the tree phytohormone-mediated defenses. Light microscopy showed that the pathogen occurred in most cell types and structures within infected E. grandis stem tissue. Notably, the fungus appeared to spread through the stem by penetrating cell wall pits. In order to understand the molecular interaction between these organisms and predict putative pathogenicity mechanisms of C. austroafricana, fungal gene expression was studied in vitro and in planta. Fungal genes associated with cell wall degradation, carbohydrate metabolism and phytohormone manipulation were expressed in planta by C. austroafricana. These genes could be involved in fungal spread by facilitating cell wall pit degradation and manipulating phytohormone mediated defense in each host environment, respectively. Specifically, the in planta expression of an ent-kaurene oxidase and salicylate hydroxylase in C. austroafricana suggests putative mechanisms by which the pathogen can modulate the phytohormone-mediated defenses of the host. These mechanisms have been reported in herbaceous plant–pathogen interactions, supporting the notion that these aspects of the interaction are similar in a woody species. This study highlights ent-kaurene oxidase and salicylate hydroxylase as candidates for further functional characterization.