Fusarium diversity from the Golden Gate Highlands National Park.

Members from the genus Fusarium can infect a broad range of plants and threaten agricultural and horticultural production. Studies on the diversity of Fusarium occurring in natural ecosystems have received less attention than the better known phytopathogenic members of the genus. This study identified Fusarium species from soils with low anthropogenic disturbance found in the Golden Gate Highlands National Park (GGHNP), a part of the Drakensberg system in South Africa. Selective techniques were implemented to obtain 257 individual isolates from the selected soil samples for which the translation elongation factor 1α (tef-1α) gene region was sequenced and compared against the Fusarium MLST and FUSARIUM-ID databases. Phylogenetic analyses, based on maximum likelihood and Bayesian inference, were used to determine species diversity in relation to reference isolates. Species level identifications were made within three of the seven species complexes and identified F. brachygibbosum, F. sporotrichioides, F. andiyazi, and F. gaditjirri based on the FUSARIUM-ID database, with F. transvaalense and F. lyarnte identified against the Fusarium MLST database. This indicated highly diverse populations of Fusarium from soils with low anthropogenic disturbance from the Afromontane grassland region found in mountain ranges.

Historical Development of the Puccinia triticina Population in South Africa.

In contrast to many other countries, the virulence and genetic diversity of the South African Puccinia triticina population before 1980 is unknown, because of the absence of regular and systematic race analysis data and viable rust cultures. Herbarium specimens housed at the National Collection of Fungi, Biosystematics Unit, Plant Health and Protection, Agricultural Research Council, Pretoria, South Africa (SA), provided the opportunity to investigate the genetic development of the population using isolates collected between 1906 and 2010. Five subpopulations that survived between 21 and 82 years in the field were found. While three of these could represent the original races that entered SA during European settlement, two appear to be recent exotic introductions into SA, most probably from other African countries. The demise of the three oldest subpopulations might be from the release of resistant wheat cultivars. The population is clonal, where new virulence develops through single step mutations and selection for virulence. Although a possible case of somatic hybridization was found, sexual reproduction appears to be absent in SA. This study confirmed the importance of annual surveys in SA and its neighboring countries for the timely detection of new virulent races that could threaten wheat production in SA.

Genetic Diversity of the Fusarium oxysporum Complex Isolated from the Grassland Biome of South Africa.

The genetic diversity of pathogenic members of the Fusarium oxysporum species complex (FOSC) has been intensively studied worldwide, yet strains occurring in native soils with low anthropogenic disturbance remain poorly understood. This study focused on 355 F. oxysporum isolates from soils with low anthropogenic activity obtained from the grassland biome of South Africa. Analysis of the translation elongation factor 1-alpha (tef-1α) gene revealed high levels of sequence type diversity within the soil population in comparison with the global dataset. Phylogenetic relationships of the South African isolates revealed that four nested within FOSC clade 1. This is the first report of members of the basal clade recovered from ecosystems with low anthropogenic disturbance from Sub-Saharan Africa. The remaining strains nested within clades 2 to 5. This study contributes significantly to our understanding of the distribution of the FOSC in natural systems as we show that FOSC populations in the South African grassland biome are genetically diverse. This fills in our knowledge gap because previous studies reported only on the occurrence and diversity of the FOSC isolated from plant debris in South Africa. This is the first comprehensive survey of fusaria from grassland soils with low anthropogenic disturbance in South Africa.

Genome-wide association analysis of Russian wheat aphid (Diuraphis noxia) resistance in Dn4 derived wheat lines evaluated in South Africa.

Russian wheat aphid (RWA; Diuraphis noxia Kurdjumov) resistance on the 1D chromosome of wheat has been the subject of intensive research. Conversely, the deployment of the Dn4 derived RWA resistant varieties diminished in recent years due to the overcoming of the resistance it imparts in the United States of America. However, this resistance has not been deployed in South Africa despite reports that Dn4 containing genotypes exhibited varying levels of resistance against the South African RWA biotypes. It is possible that there may be certain genetic differences within breeding lines or cultivars that influence the expression of resistance. The aim of this study was to identify single nucleotide polymorphism (SNP) markers associated with resistance to South African RWA biotypes. A panel of thirty-two wheat lines were phenotyped for RWA resistance using four South African RWA biotypes and a total of 181 samples were genotyped using the Illumina 9K SNP wheat chip. A genome wide association study using 7598 polymorphic SNPs showed that the population was clustered into two distinct subpopulations. Twenty-seven marker trait associations (MTA) were identified with an average linkage disequilibrium of 0.38 at 10 Mbp. Four of these markers were highly significant and three correlated with previously reported quantitative trait loci linked to RWA resistance in wheat. Twenty putative genes were annotated using the IWGSC RefSeq, three of which are linked to plant defence responses. This study identified novel chromosomal regions that contribute to RWA resistance and contributes to unravelling the complex genetics that control RWA resistance in wheat.

Wheat Argonaute 5 Functions in Aphid-Plant Interaction.

Aphids feeding on plants experience similar responses to pathogens due to the prolonged and intimate contact with the plant. Diuraphis noxia is an economically important aphid pest on wheat that exhibits such an interaction. Studies on small RNA (sRNA) that regulate genes imparting resistance to wheat against D. noxia have predicted an Argonaute 5 (TaAGO5) gene as possible role player in the resistance response. Functional characterization revealed that TaAGO5 is crucial in regulating the response to infestation by D. noxia. Knockdown of TaAGO5 by 22% in D. noxia resistant wheat resulted in a completely susceptible phenotype. The fecundity and stress levels of D. noxia feeding on these silenced plants were similar to aphids feeding on the susceptible controls. Thus, TaAGO5 is crucial in the defense response by wheat plants during aphid feeding and this is similar to Nicotiana benthaminia plants experiencing arthropod herbivory. Additionally, TaAGO5 was differentially regulated by the Barley mosaic virus (BMV) used in the functional characterization. This provides evidence that TaAGO5 could play a role during virus infection of wheat. The role of AGO5 proteins in plant responses to arthropod herbivory and virus infection is known for dicotyledonous plants. Here, we present data that indicate that this role of TaAGO5 is conserved in wheat and possibly for monocotyledonous plants. These observations extend our knowledge on the roles of AGO proteins in plant resistance.

Characterisation of members of the Fusarium incarnatum-equiseti species complex from undisturbed soils in South Africa.

The genus Fusarium hosts a large number of economically significant phytopathogens with a global distribution. Surprisingly, only a limited number of studies have tried to identify the natural distribution of members of this genus in undisturbed soils. Members of the Fusarium incarnatum-equiseti species complex (FIESC) are increasingly associated with plant disease, and human and animal health problems. Recently, an outbreak of kikuyu poisoning of cattle was attributed to the F. incarnatum-equiseti species complex. Thus, it is of importance to identify the natural distribution of members of the FIESC from the environment. The aim of this study was to use the phylogenetic signal within the TEF 1α gene region to characterise 54 F. incarnatum-equiseti isolates obtained from undisturbed soils from the grassland biome of South Africa. These isolates were further compared with members of the FIESC previously associated with kikuyu poisoning of cattle. The phylogenetic analysis indicated a high level of variation within this species complex. Several members were closely related to isolates implicated in the death of cattle from infected kikuyu grass.

Silencing of a Unique Integrated Domain Nucleotide-Binding Leucine-Rich Repeat Gene in Wheat Abolishes Diuraphis noxia Resistance.

Plants respond in a similar manner to aphid feeding as to pathogen attack. Diuraphis noxia is a specialist aphid, feeding only on selected grasses that include wheat, barley, and oats. The wheat-Diuraphis noxia interaction is characterized by responses very similar to those seen in wheat-pathogen interactions with none of the underlying resistance pathways and genes characterized yet. From wheat harboring the Dn1 resistance gene, we have identified a nucleotide-binding leucine-rich repeat (NLR) gene containing two integrated domains (IDs). These are three C-terminus ankyrin repeat domains and an N-terminus WRKY domain. The NLR core of the gene can be traced through speciation events within the grass family, with a recent WRKY domain integration that is Triticum-specific. Virus-induced gene silencing of the gene in a resistant wheat line resulted in the abolishment of the resistance response and induced a highly susceptible phenotype. Silenced plants supported a higher number of aphids, similar to the susceptible near-isogenic line (NIL), and the intrinsic rate of increase of the aphids matched that of aphids feeding on the susceptible NIL. The presence of the gene is necessary for Dn1 resistance and we have named the gene Associated with Dn resistance 1 (Adnr1) to reflect this function.

Isolation of Early-Responsive MicroRNA From Diuraphis noxia (Hemiptera: Aphididae)-Resistant Wheat.

The Russian wheat aphid (Diuraphis noxia Kurdjumov) is an economically important pest of small grains in many countries. The past decades have seen the deployment of resistance-carrying wheat (Triticum aestivum L.) cultivars to control D. noxia. However, the emergence of resistance-breaking biotypes is negating this strategy. The role that noncoding RNA (ncRNA) molecules play in the wheat-D. noxia interaction has not been studied to date. This study aimed to isolate differentially regulated microRNA from a resistant and susceptible near-isogenic wheat line after aphid infestation. Twenty-seven identified miRNA were mostly related to stress-linked miRNA, and their predicted targets were linked with known D. noxia-feeding regulated proteins. These included transcription factors, signaling proteins, carbohydrate metabolism, and disease resistance pathways. Gene expression of three putative miRNAs and a predicted nucleotide-binding leucine-rich repeat gene with an identified miRNA target site in the NB-ARC domain displayed differential regulation between the resistant and susceptible plants. This study marks the initial investigation into understanding the role of ncRNA in a D. noxia-resistant wheat line after infestation and reports a correlation between a miRNA and its putative target for this interaction.

Involvement of cathepsin B in the plant disease resistance hypersensitive response.

A diverse range of plant proteases are implicated in pathogen perception and in subsequent signalling and execution of disease resistance. We demonstrate, using protease inhibitors and virus-induced gene silencing (VIGS), that the plant papain cysteine protease cathepsin B is required for the disease resistance hypersensitive response (HR). VIGS of cathepsin B prevented programmed cell death (PCD) and compromised disease resistance induced by two distinct non-host bacterial pathogens. It also suppressed the HR triggered by transient co-expression of potato R3a and Phytophthora infestans Avr3a genes. However, VIGS of cathepsin B did not compromise HR following recognition of Cladosporium fulvum AVR4 by tomato Cf-4, indicating that plant PCD can be independent of cathepsin B. The non-host HR to Erwinia amylovora was accompanied by a transient increase in cathepsin B transcript level and enzymatic activity and induction of the HR marker gene Hsr203. VIGS of cathepsin B significantly reduced the induction of Hsr203 following E. amylovora challenge, further demonstrating a role for this protease in PCD. Whereas cathepsin B is often relocalized from the lysosome to the cytosol during animal PCD, plant cathepsin B is secreted into the apoplast, and is activated upon secretion in the absence of pathogen challenge.

A novel non-protein-coding infection-specific gene family is clustered throughout the genome of Phytophthora infestans.

Phytophthora infestans is the cause of late blight, a devastating and re-emerging disease of potato. Significant advances have been made in understanding the biology of P. infestans, and in the development of molecular tools to study this oomycete. Nevertheless, little is known about the molecular bases of the establishment or development of disease in this hemibiotrophic pathogen. Suppression subtractive hybridization (SSH) was used to generate cDNA enriched for sequences upregulated during potato infection. To identify pathogen-derived cDNAs, and eliminate host sequences from further study, SSH cDNA was hybridized to a P. infestans bacterial artificial chromosome library. A new gene family was identified called Pinci1, comprising more than 400 members arranged in clusters of up to nine copies throughout the P. infestans draft genome sequence. Real-time RT-PCR was used to quantify the expression of five classes of transcript within the family, relative to the constitutively expressed PiactA gene, and it revealed them to be significantly upregulated from 12 to 33 h post-inoculation, a period defining the biotrophic phase of infection. Computational analysis of sequences suggested that transcripts were non-protein coding, and this was confirmed by transient expression of FLAG-tagged ORFs in P. infestans.

Is photosynthetic transcriptional regulation in Triticum aestivum L. cv. 'TugelaDN' a contributing factor for tolerance to Diuraphis noxia (Homoptera: Aphididae)?

Diuraphis noxia (Russian wheat aphid, RWA) is a major pest on wheat in South Africa and most other wheat growing countries. Being a probing-sucking insect, RWAs insert their stylets into the phloem sieve elements and feed on the phloem sap. This feeding causes necrotic lesions in resistant varieties, or decoloration of leaves and death in susceptible varieties. In an effort to broaden our understanding on the response of the plant to RWA feeding, we synthesized and analyzed expressed sequence tags (ESTs) from suppression subtractive hybridization (SSH) libraries. These libraries were constructed using near isogenic wheat lines susceptible "Tugela" and resistant "TugelaDN" (Dn1) to RWA, as well as accession lines PI137739 (Dn1) and PI294994 (Dn5). Analysis of 200 ESTs from the libraries revealed the involvement of transcripts encoding genes involved in cell maintenance, growth and regulation, plant defense and signaling, photosynthesis and energy production, and of unknown function. A selection of these ESTs, in combination with clones obtained from other sources, were used on a custom array to study the expression profiles of 256 candidate wheat sequences putatively involved in plant defense against RWA. The selected sequences included wheat genomic clones with putative nucleotide binding site (NBS) motifs, rapid amplification of cDNA ends PCR (RACE-PCR), and cDNA clones from RWA induced libraries. Genomic banana and flax clones that were obtained using representative difference analysis (RDA), and suspected to be involved in abiotic stress responses, were also spotted onto the microarray slides. The spotted custom arrays were then hybridized against cDNA isolated from a resistant cultivar "TugelaDN" on 0, 2, 5, and 8 days after infestation, post-labeled with Cy3- or Cy5-fluorescent dyes. The subsequent expression profiling using DNA microarray, RT-PCR, and Northern Blot analysis identified 29 transcripts associated with the feeding response. These transcripts encoded proteins functioning in direct defense and signaling, oxidative burst, cell wall degradation, cell maintenance, photosynthesis, and energy production. Results indicate that plants co-ordinately regulate gene expression when attacked by RWA. It is hypothesized that the NBS-LRR proteins are important in receptor recognition and signaling, which enable the plant to overcome the stresses inflicted by RWA feeding. It is further suggested that the ability to maintain photosynthetic function with resultant energy production is one of the determining factors ensuring the survival of the resistant varieties when coping with the RWA feeding.

An ancestral oomycete locus contains late blight avirulence gene Avr3a, encoding a protein that is recognized in the host cytoplasm.

The oomycete Phytophthora infestans causes late blight, the potato disease that precipitated the Irish famines in 1846 and 1847. It represents a reemerging threat to potato production and is one of >70 species that are arguably the most devastating pathogens of dicotyledonous plants. Nevertheless, little is known about the molecular bases of pathogenicity in these algae-like organisms or of avirulence molecules that are perceived by host defenses. Disease resistance alleles, products of which recognize corresponding avirulence molecules in the pathogen, have been introgressed into the cultivated potato from a wild species, Solanum demissum, and R1 and R3a have been identified. We used association genetics to identify Avr3a and show that it encodes a protein that is recognized in the host cytoplasm, where it triggers R3a-dependent cell death. Avr3a resides in a region of the P. infestans genome that is colinear with the locus containing avirulence gene ATR1(NdWsB) in Hyaloperonospora parasitica, an oomycete pathogen of Arabidopsis. Remarkably, distances between conserved genes in these avirulence loci were often similar, despite intervening genomic variation. We suggest that Avr3a has undergone gene duplication and that an allele evading recognition by R3a arose under positive selection.

Profiling and quantifying differential gene transcription in Phytophthora infestans prior to and during the early stages of potato infection.

Phytophthora infestans, the causal agent of potato and tomato late blight, produces several different cell types prior to and during the early stages of potato infection. All of these cell types can be easily generated and studied in the absence of the host plant and so form the basis for developmental stage-specific gene discovery. We have used amplified fragment length polymorphism (AFLP)-based mRNA fingerprinting (cDNA-AFLP) to identify 64 transcripts that appeared to be up-regulated in germinating cysts but not in vegetative mycelium. These transcripts included representatives of most major classes of heat shock proteins: hsp60, hsp70, hsp90, and hsp100. Real-time RT-PCR was used to quantify the expression of 18 transcripts originating from germinating cysts, relative to the constitutively expressed actB gene, in vegetative mycelium, germinating cysts, and at three time-points post-inoculation of potato cultivar Bintje (15, 48, and 72h). All of the transcripts were up-regulated in germinating cysts, and 12, including hsp70, hsp80-2, and hsp90, were found also to be up-regulated in planta. This is the first report of the application of real-time RT-PCR to the relative quantification of plant pathogen gene expression during the early stages of infection.