Ceratocystis Albifundus Research Articles

Transferring an Agrobacterium‐mediated transformation protocol across eight genera in the Ceratocystidaceae

AbstractEstablishing a transformation system is an essential first step for functional studies. Numerous transformation systems have been developed and optimized for filamentous ascomycetes, including Agrobacterium‐mediated transformation. Recently, such a system was developed for the African tree pathogen Ceratocystis albifundus. This fungus is part of the family Ceratocystidaceae, which includes many tree and plant pathogens of economic importance. Despite the many advantages associated with Agrobacterium‐mediated transformation, establishing and optimizing this system within a species are extremely arduous due to the many species‐specific parameters that must be optimized. This study aimed to apply the existing C. albifundus Agrobacterium‐mediated transformation protocol to eight diverse Ceratocystidaceae species from eight different genera. Minor adjustments were made to the already established protocol to increase transformant yield before it was used to successfully produce transformants for six of the eight targeted species without the need for any further species‐specific optimizations. Although the number of transformants obtained was less than for C. albifundus, this study proves that protocols for Agrobacterium‐mediated transformation might be more transferable than previously thought. It also provides a useful benchmark for future transformation studies in the Ceratocystidaceae and will make species‐specific optimization easier.

Quantification of Outcrossing Events in Haploid Fungi Using Microsatellite Markers.

Species in genera of the fungal family Ceratocystidaceae are known to have different mating strategies, including heterothallism and homothallism. Of these, species of Ceratocystis, typified by the pathogen Ceratocystis fimbriata all undergo unidirectional mating-type switching. This implies that the pathogens possess the ability to self, but also to undergo sexual outcrossing between isolates of different mating types. In this study, we extended the recently developed microsatellite-based technique to determine the extent to which outcrossing occurs in ascospore masses of haploid fungi to two field collections of Ceratocystis albifundus. In this way, the role of reproductive strategies in shaping population structure and diversity could be better understood. Results showed that a high frequency of outcrossing occurs in isolates of the pathogen from both non-native and native areas. This explains the high level of genetic diversity previously observed in this population despite the fact that this pathogen has the ability to self.

Agrobacterium-mediated transformation of Ceratocystis albifundus

Functional association between genomic loci and specific biological traits remains lacking in many fungi, including the African tree pathogen Ceratocystis albifundus. This is mainly because of the absence of suitable transformation systems for allowing genetic manipulation of this and other fungi. Here, we present an optimized protocol for Agrobacterium tumefaciens-mediated transformation of C. albifundus. Strain AGL-1 of A. tumefaciens and four binary T-DNA vectors (conferring hygromycin B or geneticin resistance and/or expressing the green fluorescent protein [GFP]) were used for transforming germinated conidia of three isolates of C. albifundus. Stable expression of these T-DNA-encoded traits was confirmed through sequential sub-culturing of fungal transformants on selective and non-selective media and by using PCR and sequence analysis. Single-copy integration of the respective T-DNAs into the genomes of these fungi was confirmed using Southern hybridization analysis. The range of experimental parameters determined and optimised included: (i) concentrations of hygromycin B and geneticin required for inhibiting growth of the wild type fungus and (ii) the dependence of transformation on acetosyringone for inducing the bacterium’s virulence genes, as well as (iii) the duration of fungus-bacterium co-cultivation periods and (iv) the concentrations of fungal conidia and bacterial cells used for the latter. The system developed in this study is stable with a high-efficiency, yielding up to 400 transformants per 106 conidia. This is the first report of a transformation protocol for C. albifundus and its availability will be invaluable for functional studies in this important fungus.

Genomic analysis of the aggressive tree pathogen Ceratocystis albifundus

The overall goal of this study was to determine whether the genome of an important plant pathogen in Africa, Ceratocystis albifundus, is structured into subgenomic compartments, and if so, to establish how these compartments are distributed across the genome. For this purpose, the publicly available genome of C. albifundus was complemented with the genome sequences for four additional isolates using the Illumina HiSeq platform. In addition, a reference genome for one of the individuals was assembled using both PacBio and Illumina HiSeq technologies. Our results showed a high degree of synteny between the five genomes, although several regions lacked detectable long-range synteny. These regions were associated with the presence of accessory genes, lower genetic similarity, variation in read-map depth, as well as transposable elements and genes associated with host-pathogen interactions (e.g. effectors and CAZymes). Such patterns are regarded as hallmarks of accelerated evolution, particularly of accessory subgenomic compartments in fungal pathogens. Our findings thus showed that the genome of C. albifundus is made-up of core and accessory subgenomic compartments, which is an important step towards characterizing its pangenome. This study also highlights the value of comparative genomics for understanding mechanisms that may underly and influence the biology and evolution of pathogens.

A microsatellite-based identification tool used to confirm vector association in a fungal tree pathogen

Many fungi live in close association with insects, and some are specifically vectored by them. One of the best examples is found in the so-called Ophiostomatoid fungi, including species of Ceratocystis and other genera in the Ceratocystidaceae. Our understanding of vectorship in these fungi is based predominantly on either their frequency of isolation from insects or the success with which these fungi are isolated from their insect vectors. The fact that Ceratocystis species mostly have casual as opposed to highly specific relationships with their insect vectors makes it difficult to prove insect vector relations. In order to provide unambiguous support for Ceratocystis species being vectored by insects, we interrogated whether genotypes of the tree pathogen, Ceratocystis albifundus, would be shared between isolates retrieved from either infected trees or nitidulid beetles. Ceratocystis albifundus isolates were collected from nitidulid beetles and from naturally occurring wounds on trees in the Kruger National Park (KNP) in South Africa. The genotypes of these isolates were then determined using eight microsatellite markers, and they were compared using a haploid network analyses. A high frequency of Multi-Locus Haplotypes (MLHs) derived from nitidulid beetles was found to be shared with those from wounded trees across the KNP. This provides robust support showing that nitidulid beetles play an important role in the dispersal of C. albifundus in the KNP.

Non-Mendelian segregation influences the infection biology and genetic structure of the African tree pathogen Ceratocystis albifundus

The African fungal tree pathogen, Ceratocystis albifundus, undergoes uni-directional mating type switching, giving rise to either self-fertile or self-sterile progeny. Self-sterile isolates lack the MAT1-2-1 gene and have reduced fitness such as slower growth and reduced pathogenicity, relative to self-fertile isolates. While it has been hypothesized that there is a 1:1 ratio of self-fertile to self-sterile ascospore progeny in relatives of C. albifundus, some studies have reported a significant bias in this ratio. This could be due to the fact that either fewer self-sterile ascospores are produced or that self-sterile ascospores have low viability. We quantified the percentage of self-sterile and self-fertile ascospores from ascospore masses in C. albifundus using real-time PCR. Primers were designed to distinguish between spores that contained the MAT1-2-1 gene and those where this gene had been deleted. A significant bias towards the self-fertile mating type was observed in all single ascospore masses taken from sexual structures produced in haploid-selfed cultures. The same result was observed from a disease outbreak situation in an intensively managed field of cultivated native trees, and this was coupled with very low population diversity in the pathogen. This was in contrast to the results obtained from ascospore masses taken from the crosses performed under laboratory conditions or ascomata on native trees in a non-disease situation, where either self-fertile or self-sterile ascospores were dominant. The results suggest that reproductive strategies play a significant role in the infection biology and genetic structure of C. albifundus populations.

Contrasting carbon metabolism in saprotrophic and pathogenic microascalean fungi from Protea trees

Abstract Protea-associated Knoxdaviesia species grow on decaying inflorescences, yet are closely related to plant pathogens such as Ceratocystis albifundus. C. albifundus also infects Protea, but occupies a distinct niche. We investigated substrate utilization in two Knoxdaviesia saprotrophs, a generalist and a specialist, and the pathogen C. albifundus by integrating phenome and whole-genome data. On shared substrates, the generalist grew slightly better than its specialist counterpart, alluding to how it has maintained its Protea host range. C. albifundus grew on few substrates and had limited cell wall-degrading enzymes. It did not utilize sucrose, but may prefer soluble oligosaccharides. Nectar monosaccharides are likely important carbon sources for early colonizing Knoxdaviesia species. Once the inflorescence ages, they could switch to degrading cell wall components. C. albifundus likely uses its limited cell wall-degrading arsenal to gain access to plant cells and exploit internal resources. Overall, carbon metabolism and gene content in three related fungi reflected their ecological adaptations.

Ecology and population structure of a tree wound-infecting fungus in a native South African forest environment

Ceratocystis tsitsikammensis was first isolated from bark harvesting wounds on two indigenous tree species in the Afromontane forests of the Western Cape Province of South Africa. Inoculation studies indicated that it is a potential pathogen of native Rapanea melanophloeos trees. In this study, we investigated the distribution, ecology and biology of C. tsitsikammensis in the Garden Route National Park of South Africa. Isolates were obtained from wounds on R. melanophloeos, three non-native hosts as well as from nitidulid and staphylinid beetles visiting wounds on these trees. The genetic diversity and population biology of the fungus was examined using microsatellite markers. Its mating strategy was also determined by amplifying its mating type genes and the fungus was shown to be homothallic. Despite the homothallic nature of the fungus, high levels of random mating and absence of genetic structure was found in the investigated population, suggesting a strong effect of gene flow, probably linked to insect dispersal. The gene diversity of C. tsitsikammensis was similar to that of a related fungus, Ceratocystis albifundus, that is known to be native in Africa. This, together with the fact that C. tsitiskamensis is not known elsewhere, within or outside South Africa, suggests that it is native and endemic to the Cape Afromontane region.

The genetic landscape of Ceratocystis albifundus populations in South Africa reveals a recent fungal introduction event

Geographical range expansion or host shifts is amongst the various evolutionary forces that underlie numerous emerging diseases caused by fungal pathogens. In this regard, Ceratocystis albifundus, the causal agent of a serious wilt disease of Acacia mearnsii trees in Africa, was recently identified killing cultivated Protea cynaroides in the Western Cape (WC) Province of South Africa. Protea cynaroides is an important native plant in the area and a key component of the Cape Floristic Region. The appearance of this new disease outbreak, together with isolates of C. albifundus from natural ecosystems as well as plantations of nonnative trees, provided an opportunity to consider questions relating to the possible origin and movement of the pathogen in South Africa. Ten microsatellite markers were used to determine the genetic diversity, population structure, and possible gene flow in a collection of 193 C. albifundus isolates. All populations, other than those from the WC, showed high levels of genetic diversity. An intermediate level of gene flow was found amongst populations of the pathogen. The results suggest that a limited number of individuals have recently been introduced into the WC, resulting in a novel disease problem in the area.

Variation in growth rates and aggressiveness of naturally occurring self‐fertile and self‐sterile isolates of the wilt pathogen Ceratocystis albifundus

Ceratocystis albifundus is the most important fungal pathogen of black wattle (Acacia mearnsii) grown in plantations in southern and eastern Africa. It is a homothallic fungus but also undergoes unidirectional mating type switching. As a result, the ascospore progeny can be either self‐fertile or self‐sterile. The only apparent difference between these mating types is the deletion of the MAT1‐2‐1 gene in self‐sterile isolates. There is some evidence suggesting that self‐sterile isolates grow more slowly than self‐fertile isolates, but this has not been tested rigorously. The aim of this study was to determine whether self‐sterile isolates are less fit by examining growth rate, relative germination rate and pathogenicity. Five self‐sterile isolates were generated from each of five self‐fertile isolates of C. albifundus and these 30 isolates were compared. The results showed that the self‐sterile isolates grew consistently slower and were less pathogenic than the self‐fertile isolates. The germination ratio of self‐fertile to self‐sterile isolates from single ascospores collected from the ascomata of five self‐fertile isolates was on average 7:3. This could be a consequence of the self‐sterile isolates having a lower germination rate. This observation, and the lower growth and pathogenicity levels, suggests that self‐sterile isolates are not likely to compete effectively in nature, raising intriguing questions regarding their role and value to C. albifundus and other fungi having a similar mating system.

IMA Genome-F 3: Draft genomes of Amanita jacksonii, Ceratocystis albifundus, Fusarium circinatum, Huntiella omanensis, Leptographium procerum, Rutstroemia sydowiana, and Sclerotinia echinophila.

The genomes of fungi provide an important resource to resolve issues pertaining to their taxonomy, biology, and evolution. The genomes of Amanita jacksonii, Ceratocystis albifundus, a Fusarium circinatum variant, Huntiella omanensis, Leptographium procerum, Sclerotinia echinophila, and Rutstroemia sydowiana are presented in this genome announcement. These seven genomes are from a number of fungal pathogens and economically important species. The genome sizes range from 27 Mb in the case of Ceratocystis albifundus to 51.9 Mb for Rutstroemia sydowiana. The latter also encodes for a predicted 17350 genes, more than double that of Ceratocystis albifundus. These genomes will add to the growing body of knowledge of these fungi and provide a value resource to researchers studying these fungi.

Molecular phylogenetic analyses reveal three new Ceratocystis species and provide evidence for geographic differentiation of the genus in Africa

The emergence of wattle wilt disease on non-native Acacia mearnsii trees in Africa, caused by the indigenous fungus Ceratocystis albifundus, has highlighted a need to better understand the diversity, ecology and distribution of Ceratocystis species in natural African environments. In this study we applied phylogenetic inference to identify and characterize isolates of Ceratocystis collected in a natural savanna ecosystem in South Africa. Three new species were recognized and are described as C. cryptoformis sp. nov. in the C. moniliformis complex, as well as C. thulamelensis sp. nov. and C. zambeziensis sp. nov., both residing in the C. fimbriata complex. Incorporating the new species into global phylogenies of Ceratocystis provided insights into the patterns of evolution and biogeography of this group of fungi. Notably, the African continent was identified as an important centre of diversification of Ceratocystis spp., from which several lineages of these fungi were shown to have radiated.

Factors influencing infection of Acacia mearnsii by the wilt pathogen Ceratocystis albifundus in South Africa

SummaryIt is well known that species of Ceratocystis, which cause canker and wilt diseases on trees, require wounds for infection. In this regard, a number of physical and environmental factors influence the success of wound colonization by these fungi. The aim of this study was to consider the influence of wound age, stem diameter and colonization of wounds by Ophiostoma quercus on the success of infection by Ceratocystis albifundus, an important wilt pathogen of Acacia mearnsii in southern and eastern Africa, under field conditions. This was performed by doing controlled inoculations on Acacia mearnsii trees, of different diameters, with C. albifundus at different time intervals after wounding and after pre‐inoculation of wounds with O. quercus at the time of wounding. The success of infection by C. albifundus decreased significantly from 8 h after wounding and was higher on stems of greater diameter. Pre‐infection of wounds by O. quercus reduced the infection success of C. albifundus. The overall results showed that managing wounds created during forestry operations by treating wounds with naturally occurring, non‐virulent fungi, such as O. quercus, could reduce the prevalence of infections by C. albifundus.

Occurrence of the wattle wilt pathogen, Ceratocystis albifundus on native South African trees

Summary Ceratocystis albifundus causes the disease known as wattle wilt of non‐native Acacia mearnsii trees in South Africa, Uganda and Kenya. Infection results in rapid wilt and death of susceptible trees and stem cankers on more tolerant trees. It has been suggested that C. albifundus is indigenous to southern Africa, possibly having spread from native Protea spp. to non‐native A. mearnsii and A. decurrens trees. Although C. albifundus has been collected from Protea spp., these reports are based on limited records for which only aged herbarium specimens exist. During surveys of wound‐infecting fungi on native tree species in South Africa, a fungus resembling C. albifundus was collected from Protea gaguedi, Acacia caffra, Burkea africana, Combretum molle, C. zeyheri, Faurea saligna, Ochna pulchra, Ozoroa paniculosa and Terminalia sericea. The identity of the fungus was confirmed as C. albifundus, using comparisons of DNA sequence data for the ITS and 5.8S gene of the rRNA operon. In pathogenicity trials, lesions were produced on C. molle and A. caffra, with some trees beginning to die at the termination of the experiment. This study represents the first report of C. albifundus from native tree species in South Africa and provides unequivocal evidence that the fungus occurs naturally on native Protea spp. The wide host range of C. albifundus, as well as its abundance on these indigenous hosts lends further support to the view that it is a native African pathogen.

Comparison of populations of the wilt pathogen Ceratocystis albifundus in South Africa and Uganda

Ceratocystis albifundus is an important fungal pathogen of Acacia mearnsii trees in South Africa. In a previous study, a high level of gene diversity was demonstrated in a South African population of C. albifundus. This, together with the occurrence of the pathogen on native Protea species and its exclusive occurrence in South Africa, led to the hypothesis that C. albifundus is probably native to that country. More recently, C. albifundus has been reported from A. mearnsii in south‐western Uganda. The aim of this study was to compare the populations of C. albifundus from Uganda and South Africa based on genetic diversity, population structure and possible gene flow. This was achieved using codominant microsatellite markers developed for the closely related species Ceratocystis fimbriata. Available isolates for comparison were from six different areas of South Africa and six jungle stands in Uganda. Eight of the 11 available markers amplified loci in C. albifundus. Gene diversity was higher in the Ugandan population, but genotypic diversity was greater for the South African isolates. There were no common genotypes between the two populations and they shared only 22% of the total alleles. The populations were genetically isolated from each other and highly substructured within. There was no association between isolates collected from the same geographic locations, and gene flow between the two populations was low. Results suggest that C. albifundus was probably not introduced into Uganda from South Africa but rather that an ancestral population, yet to be discovered, is the source of both populations.