Molecular markers delimit cryptic species in Ceratocystis sensu stricto

Ceratocystis sensu stricto is a genus of closely related fungi that are mostly plant pathogens. Morphological variation between species in this genus is limited and species delineation is strongly reliant on phylogenetic inference. Primary support for many of the species is based on the ITS region which, on its own, can be used to delineate all species described in the genus. However, the recent discovery of two ITS types in a single isolate of Ceratocystis questions the use of this marker in taxonomic studies. The aim of this study was to consider the potential use of alternative gene regions to support the species boundaries in this genus. The phylogenetic value of the βT 1 and EF 1-α gene regions, generally used in combination with ITS, were re-evaluated and compared to five single copy protein coding genes (CAL, RPBII, MS204, FG1093 and Mcm7). As an alternative approach, genome-wide single nucleotide polymorphisms (SNPs) were identified and evaluated as diagnostic markers to distinguish between the species. Fifteen species residing in Ceratocystis were used in this study. None of the protein coding genes could be used to distinguish all species but a combination of the βT 1, MS204 and RPBII gene regions resolved 11 of the 15 described species. Unique SNP markers were identified for 13 of the species and these provided significant additional support for most of the established taxon boundaries. Other than ITS, none of the markers tested could distinguish between C. acaciivora and C. manginecans and these species are, therefore, reduced to synonymy with the name C. manginecans being retained. Results of this study also revealed the likely existence of additional species in Ceratocystis.Tree Protection Co-operative Programme (TPCP), the National Research Foundation (NRF) as well as the Genomics Research Institute (GRI) at the University of Pretoria. Also the Thuthuka Foundation (grant no 80670) .http://link.springer.comjournal/11557hb201

A possible centre of diversity in South East Asia for the tree pathogen, Ceratocystis manginecans

The fungal pathogen, Ceratocystis manginecans, has caused serious canker and wilt disease on Mangifera indica (mango), on legume tree species in Oman and Pakistan and on Acacia spp. in Indonesia. A Ceratocystis species, with similar morphology to C. manginecans, has recently been reported in Vietnam, causing severe disease of Acacia trees. Previous population genetic studies on isolates from M. indica in Oman and Pakistan have shown that the pathogen represents a single clonal haplotype, indicative of an introduced pathogen. The aim of this study was to investigate the genetic diversity and population structure of 160 C. manginecans isolates, from four host-associated populations from Oman, Pakistan, Indonesia and Vietnam. This was done by applying a combination of 14 previously developed microsatellite markers and a new set, designed in this study from two different C. manginecans genomes. Sequence data confirmed that the isolates in Vietnam are the same species as those in Indonesia and were thus identified as C. manginecans. Unlike the populations in Oman and Pakistan, relatively high levels of genetic variation were found for the isolates from Indonesia and Vietnam. The Vietnam population was significantly differentiated from the other populations and isolates from this area had the highest level of genetic diversity thus far encountered for the pathogen.Tree Protection Cooperative programme (TPCP) at the Forestry and Agricultural Biotechnology Institute (FABI),University of Pretoria. National Research Foundation (NRF) (grant no 80670).http://www.elsevier.com/locate/meegid2017-07-31hb2016Forestry and Agricultural Biotechnology Institute (FABI)Genetic

QTL mapping of mycelial growth and aggressiveness to distinct hosts in Ceratocystis pathogens

Some species of Ceratocystis display strong host specificity, such as C. fimbriata sensu stricto that is restricted to sweet potato (Ipomoea batatas) as host. In contrast, the closely related C. manginecans, infects Acacia mangium and Mangifera indica but is not pathogenic to I. batatas. Despite the economic importance of these fungi, knowledge regarding the genetic factors that influence their pathogenicity and host specificity is limited. A recent inheritance study, based on an interspecific cross between C. fimbriata and C. manginecans and the resultant 70 F1 progeny, confirmed that traits such as mycelial growth rate, spore production and aggressiveness on A. mangium and I. batatas are regulated by multiple genes. In the present study, a quantitative trait locus (QTL) analysis was performed to determine the genomic loci associated with these traits. All 70 progeny isolates were genotyped with SNP markers and a linkage map was constructed. The map contained 467 SNPs, distributed across nine linkage groups, with a total length of 1203 cm. Using the progeny genotypes and phenotypes, one QTL was identified on the linkage map for mycelial growth rate, one for aggressiveness to A. mangium and two for aggressiveness to I. batatas (P < 0.05). Two candidate genes, likely associated with mycelial growth rate, were identified in the QTL region. The three QTLs associated with aggressiveness to different hosts contained candidate genes involved in protein processing, detoxification and regions with effector genes and high transposable element density. The results provide a foundation for studies considering the function of genes regulating various quantitative traits in Ceratocystis.Members of the Tree Protection Cooperative Programme, based at the Forestry and Agricultural Biotechnology Institute, and the Genomics Research Institute at the University of Pretoria, as well as the National Research Foundation, South Africa (Grant UID: 89619 and 95875) and the DST/NRF SARChI chair in Fungal Genomics.http://www.elsevier.com/locate/yfgbi2020-10-01hj2020BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Growth rate is a dominant factor predicting the rhizosphere effect

The root microbiome is shaped by plant root activity, which selects specific microbial taxa from the surrounding soil. This influence on the microorganisms and soil chemistry in the immediate vicinity of the roots has been referred to as the rhizosphere effect. Understanding the traits that make bacteria successful in the rhizosphere is critical for developing sustainable agriculture solutions. In this study, we compared the growth rate potential, a complex trait that can be predicted from bacterial genome sequences, to functional traits encoded by proteins. We analyzed 84 paired rhizosphere- and soil-derived 16S rRNA gene amplicon datasets from 18 different plants and soil types, performed differential abundance analysis, and estimated growth rates for each bacterial genus. We found that bacteria with higher growth rate potential consistently dominated the rhizosphere, and this trend was confirmed in different bacterial phyla using genome sequences of 3270 bacterial isolates and 6707 metagenome-assembled genomes (MAGs) from 1121 plant- and soil-associated metagenomes. We then identified which functional traits were enriched in MAGs according to their niche or growth rate status. We found that predicted growth rate potential was the main feature for differentiating rhizosphere and soil bacteria in machine learning models, and we then analyzed the features that were important for achieving faster growth rates, which makes bacteria more competitive in the rhizosphere. As growth rate potential can be predicted from genomic data, this work has implications for understanding bacterial community assembly in the rhizosphere, where many uncultivated bacteria reside

Copiotrophs dominate rhizosphere microbiomes and growth rate potential is a major factor explaining the rhizosphere effect

The structure and function of the root microbial community is shaped by plant root activity, enriching specific microbial taxa and functions from the surrounding soil as the plant root grows. Knowledge of bacterial rhizosphere competence traits are important for predictive microbiome modeling and the development of viable bioinoculants for sustainable agriculture solutions. In this work we compared growth rate potential, a complex trait that recently became predictable from bacterial genome sequences, to functional traits encoded by proteins. We analyzed 84 paired rhizosphere- and soil-derived 16S rRNA metabarcoding datasets from 18 different plants and soil types, performed differential abundance analyses and estimated growth rates for each bacterial genus. This analysis revealed that bacteria with a high growth rate potential consistently dominated the rhizosphere. Next, we analyzed the genome sequences of 3270 bacterial isolates and 6707 MAGs from 1121 plant- and soil-associated metagenomes, confirming this trend in different bacterial phyla. We next investigated which functional traits were enriched in the rhizosphere, expanding the catalog of rhizosphere-associated traits with hundreds of new functions. When we compared the importance of different functional categories to the predicted growth rate potential using a machine learning model, we found that growth rate potential was the main feature for differentiating rhizosphere and soil bacteria, revealing the broad importance of this factor for explaining the rhizosphere effect. Together, we contribute new understanding of the bacterial traits needed for rhizosphere competence. As this trait may be inferred from (meta-) genome data, our work has implications for understanding bacterial community assembly in the rhizosphere, where many uncultivated bacteria reside

Distinguishing between cryptic species in the Ceratocystis fimbriata sensu lato species complex

Dissertation (MSc)--University of Pretoria, 2014.GeneticsMS

A detection assay to identify alternative food sources of the two-spotted stink bug, Bathycoelia distincta (Hemiptera: Pentatomidae)

The two-spotted stink bug, Bathycoelia distincta Distant (Hemiptera: Pentatomidae), is a serious pest in South African macadamia orchards. This pest is predominantly controlled using insecticides, thus alternative control methods are essential. The stink bugs arrive as adults in the orchards, during the early nut set season, but little is known about their alternative plant hosts before their arrival. The aim of this study was to develop a PCR-based metabarcoding assay to identify plant material in the gut of B. distincta. Thereafter, the persistence of plant DNA in the gut, after switching food sources, was determined by rearing the stink bugs on Zea mays L. (Cyperales: Poaceae), transferring them to Macadamia sp. and then collecting insects at different time points. As a proof of concept, the assay was tested on insects collected from commercial macadamia orchards to determine if it can identify alternative food sources. The chloroplast gene markers, trnL and trnF, were most successful for plant DNA amplification. The time trial suggested that plant material can be detected 24 h after switching to the alternate food source and one of the samples still contained Z. mays DNA after five days. Various plant species were detected from the orchard collected samples, including known food sources of other stink bugs, such as tea plants (Camellia sinensis L. (Ericales:Theaceae)) and sunflowers (Helianthus annuus L. (Asterales: Asteraceae)). This study provides the first indication of potential alternative food sources of B. distincta. The assay developed in this study can now be implemented for large-scale field surveys to contribute to future integrated pest management strategies.https://jee.oxfordjournals.org2023-01-13hj2022BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Ceratocystis wilt on Eucalyptus : first record from South Africa

Wilt and death of an E. grandis × E. urophylla variety was recently observed in the Zululand region of KwaZulu-Natal, South Africa. Symptoms on the dying trees included a streaking pattern of discolouration in the sapwood and cambium of stems and roots. A fungus resembling a Ceratocystis sp. was consistently found sporulating on diseased material and isolated from the symptomatic root and stem tissue. DNA sequence analyses of the Ceratocystis isolates, including multiple gene regions, identified the isolates as C. eucalypticola. Multiple ITS types were identified among the isolates sequenced, similar to those found in other, closely related, Ceratocystis species. Artificial inoculations under field conditions confirmed the pathogenicity of the isolates. This is the first report of a Ceratocystis sp. causing a wilt disease of Eucalyptus in South Africa.The members of the Tree Protection Co-operative Programme (TPCP), the University of Pretoria and the National Research Foundation.http://www.tandfonline.com/loi/tsfs20hj2021BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant PathologyPlant Production and Soil Scienc

Mating genes in Calonectria and evidence for a heterothallic ancestral state

The genus Calonectria includes many important plant pathogens with a wide global distribution. In order to better understand the reproductive biology of these fungi, we characterised the structure of the mating type locus and flanking genes using the genome sequences for seven Calonectria species. Primers to amplify the mating type genes in other species were also developed. PCR amplification of the mating type genes and multi-gene phylogenetic analyses were used to investigate the mating strategies and evolution of mating type in a collection of 70 Calonectria species residing in 10 Calonectria species complexes. Results showed that the organisation of the MAT locus and flanking genes is conserved. In heterothallic species, a novel MAT gene, MAT1-2-12 was identified in the MAT1-2 idiomorph; the MAT1-1 idiomorph, in most cases, contained the MAT1-1-3 gene. Neither MAT1-1-3 nor MAT1-2-12 was found in homothallic Calonectria (Ca.) hongkongensis, Ca. lateralis, Ca. pseudoturangicola and Ca. turangicola. Four different homothallic MAT locus gene arrangements were observed. Ancestral state reconstruction analysis provided evidence that the homothallic state was basal in Calonectria and this evolved from a heterothallic ancestor.The special fund for basic scientific research of State Key Laboratory of Tree Genetics and Breeding (SKLTGB) of China, the National Natural Science Foundation of China (NSFC), the National Key R&D Program of China, the National Ten-thousand Talents Program, the GuangDong Top Young Talents Program, the Genomics Research Institute and members of the Tree Protection and Cooperation Programme (TPCP), South Africa.http://www.persoonia.orgam2021BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Inheritance of phenotypic traits in the progeny of a Ceratocystis interspecific cross

Ceratocystis fimbriata is a fungal plant pathogen that causes black rot on Ipomoea batatas. Based on inoculation studies on numerous tree species, the pathogen is known to be host specific. The closely related species, Ceratocystis manginecans, causes severe wilt on a broad range of tree hosts, including Mangifera indica, Acacia mangium and other leguminous tree species. The genetic factors underlying the pathogenicity and host specificity of Ceratocystis species have rarely been investigated. In this study, an F1 population of 70 recombinant progeny from a cross between C. fimbriata and C. manginecans was generated and the inheritance of various phenotypic traits was investigated. Results showed that colony colour, growth rate, asexual spore production and aggressiveness to I. batatas and A. mangium are all quantitative traits with high levels of heritability. However, conidia production and aggressiveness appeared to be regulated by a small number of genes. No correlation could be found between aggressiveness and other phenotypic traits, suggesting that these are inherited independently. This is the first study to consider genetic inheritance of pathogenicity and host specificity in Ceratocystis species and the results will contribute, in future, to the identification of quantitative trait loci and candidate genes associated with the traits investigated.Members of the Tree Protection Co-operative programme (TPCP) at the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria as well as the National Research Foundation (NRF) (Grant UID: 89619).http://www.elsevier.com/locate/funbio2019-07-01hj2018BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant PathologyStatistic