CHARACTERISATION OF THE FACIAL ECZEMA FUNGUS ON RYEGRASS PASTURES

The following progress has been achieved with the project funded by Milk SA: Exploring the facial eczema problem in dairy cattle in the Eastern Cape of, South Africa, with a focus on the fungus Pseudopithomyces chartarum

Aim 1: To resolve the taxonomy of the genus Pseudopithomyces with focus on Pseudopithomyces chartarum and to determine which species are associated with Sporidesmin Induced Liver Disease (Facial eczema).

Objectives:

• To obtain reference cultures from culture collections (CBS and CN)
• To generate DNA sequences that target secondary identification markers                                                                                                                                                                                                                                                                                                                                                                                                                                                                            Progress: A total of 152 strains from different collections were plated and DNA was extracted for all strains. Strains were obtained from the CN collection (n=104) and CBS (n=48). A total of 940 sequences have been generated to date. These include sequences for the following gene regions: ITS (n=171), BenA (n=148), RBB2 (n=161), LSU (n=163), TEF (n=152) and GAPDH (n=145).

Aim 2: To identify, characterize, and analyse the genetic makeup of the mating type loci (MAT) in P. chartarum to better understand its reproductive biology, genetic diversity, and potential for sexual reproduction.

Objectives:

• To perform whole-genome sequencing of P. chartarum 
• To identify the mating type loci
• To compare the mating type loci sequences of P. chartarum with other fungi, particularly closely related species, to understand the evolutionary divergence.

Progress: The mating locus has been putatively identified using the published New Zealand genome (MAT1-2-1). A MAT1-1-1 locus has been identified using the genome P. chartarum strains from South Africa. Primers for the respective regions have been designed and will be tested on the strains isolated from Humansdorp. Additional genomes were obtained including P. chartarum, P. karoo and P. sacchari. The genome data have been analysed in vitro to determine whether the MAT primers designed for P. chartarum amplify MAT loci in other Pseudopithomyces species. Preliminary results show that the designed primers are unique to P. chartarum. This will be further tested in vivo for confirmation.

Aim 3: To determine whether P. chartarum is seed borne

Objectives:

• To screen native/local and export seeds for the presence of P. chartarum
• To determine whether P. chartarum has been introduced into South Africa from other countries

Progress: Previous studies showed growth of Pseudopithomyces, but other mycotoxigenic fungal genera, such as Fusarium, Cladosporium, and Alternaria, were also isolated. For the upcoming experiments, a Multiplex PCR is planned, which will enable the potential detection of P. chartarum directly from environmental seed samples. If this approach is unsuccessful, next-generation sequencing (NGS) will be done directly on the seed samples. NGS offers significant advantages for exploring the mycobiome of grass seeds compared to traditional sequencing methods.

Aim 4: To determine which Pseudopithomyces species predominate the outbreak areas in the Eastern Cape, South Africa

Objectives:

• To isolate and culture Pseudopithomyces from samples obtained from dairy farms in Humansdorp.
• To characterise Pseudopithomyces isolates making use of morphology
• To generate DNA sequences that target secondary identification markers

Progress: A total of 337 strains of P. chartarum have been collected to date. A total of 1737 sequences have been generated to date.  These include sequences for the following gene regions: ITS (n=346), BenA (n=320), RPB2 (n=74), LSU (n=330), TEF (n=333) and GAPDH (n=334). Representative sequences were shared with Dr. Bevan Wier from New Zealand, who confirmed that there is only a single Pseudopithomyces species present in the five sampling regions of this project. Dr Wier also confirmed that the species present in the Eastern Cape group phylogenetically concurs with the toxin producing species in New Zealand. 

Aim 5: Before determining the population genetics with New Zealand, the population diversity of P. chartarum strains in Humansdorp has to be determined.

Objective:

• To determine the possible origin of P. chartarum using population genetics

Progress: The mating locus has been putatively identified using the published New Zealand genome (MAT1-2-1). A MAT1-1-1 locus has been identified using the genome P. chartarum strains from South Africa. Primers for the respective regions have been designed and are being tested on the strains isolated from Humansdorp, Eastern Cape.

Aim 6: To determine the relationship between sporidesmin A concentrations and the presence of Pseudopithomyces species in the different pastures

Objective:

• To screen Pseudopithomyces isolates isolated from the pastures for sporidesmin production

Progress: Sporidesmin A has been found to be produced by 70% of the strains. Additionally, sporidesmins D and C/E were found to co-occur with sporidesmin A.

Aim 7: To explore the genome sequence of Pseudopithomyces to identify and characterize the gene cluster potentially responsible for the production of sporidesmin A toxin, a known cause of facial eczema, and to compare these findings with isolates from New Zealand. This research seeks to enhance the understanding of the Pseudopithomyces clade through comparative genomics and functional characterization of relevant biosynthetic pathway genes.

Objectives:

• To sequence the genome of Pseudopithomyces and identify putative genes involved in the biosynthesis of sporidesmin A toxin.
• To perform comparative genomic analyses between the Pseudopithomyces genome and New Zealand isolates to elucidate differences and similarities in the gene clusters responsible for toxin production.
• To describe the gene cluster associated with sporidesmin A production, determining its structure and potential regulatory mechanisms.
• To conduct functional assays on the pathway genes identified in the sporidesmin A toxin cluster to confirm their role in the synthesis of this toxin.

Progress: Two genomes of P. chartarum were successfully sequenced in collaboration with Dr. Jerome Collemare from the Westerdijk Fungal Biodiversity Centre in the Netherlands. Also, gene knock-out and transformation experiments were commenced, targeting the NRPS gene within the putatively identified sporidesmin gene cluster in collaboration with Austrian researchers, Professors Joseph Strauss and Markus Gorfer. A total of eight potential transformants containing the complete deletion construct have been identified. The transformants were PCR amplified and are being sequenced.