- Research article
- Open Access
Analysis of the Pantoea ananatis pan-genome reveals factors underlying its ability to colonize and interact with plant, insect and vertebrate hosts
© De Maayer et al.; licensee BioMed Central Ltd. 2014
- Received: 30 December 2013
- Accepted: 20 May 2014
- Published: 27 May 2014
Pantoea ananatis is found in a wide range of natural environments, including water, soil, as part of the epi- and endophytic flora of various plant hosts, and in the insect gut. Some strains have proven effective as biological control agents and plant-growth promoters, while other strains have been implicated in diseases of a broad range of plant hosts and humans. By analysing the pan-genome of eight sequenced P. ananatis strains isolated from different sources we identified factors potentially underlying its ability to colonize and interact with hosts in both the plant and animal Kingdoms.
The pan-genome of the eight compared P. ananatis strains consisted of a core genome comprised of 3,876 protein coding sequences (CDSs) and a sizeable accessory genome consisting of 1,690 CDSs. We estimate that ~106 unique CDSs would be added to the pan-genome with each additional P. ananatis genome sequenced in the future. The accessory fraction is derived mainly from integrated prophages and codes mostly for proteins of unknown function. Comparison of the translated CDSs on the P. ananatis pan-genome with the proteins encoded on all sequenced bacterial genomes currently available revealed that P. ananatis carries a number of CDSs with orthologs restricted to bacteria associated with distinct hosts, namely plant-, animal- and insect-associated bacteria. These CDSs encode proteins with putative roles in transport and metabolism of carbohydrate and amino acid substrates, adherence to host tissues, protection against plant and animal defense mechanisms and the biosynthesis of potential pathogenicity determinants including insecticidal peptides, phytotoxins and type VI secretion system effectors.
P. ananatis has an ‘open’ pan-genome typical of bacterial species that colonize several different environments. The pan-genome incorporates a large number of genes encoding proteins that may enable P. ananatis to colonize, persist in and potentially cause disease symptoms in a wide range of plant and animal hosts.
- Lateral Gene Transfer
- Core Genome
- Accessory Genome
- Average Amino Acid Identity
Pantoea ananatis is a member of the family Enterobacteriaceae and is characterised by its ubiquity in nature and its frequent association with both plant and animal hosts. It has been found in a wide array of environments including rivers, soil samples, refrigerated beef and aviation fuel tanks [1, 2]. P. ananatis is most frequently isolated from plant materials, including roots, leaves and stems of a broad range of plant hosts and exists as part of the epiphytic and endophytic flora . Since its identification as the causal agent of fruitlet rot of pineapple in the Philippines in 1928 , P. ananatis has been implicated in diseases of a wide range of host crops including maize and onion, Eucalyptus, sudangrass and honeydew melons [1, 4]. Individual isolates also appear to be capable of causing disease symptoms on a wide range of hosts. For example, strains pathogenic on rice and pineapple were demonstrated to cause blight symptoms on onion . Conversely, some P. ananatis strains have been shown to promote plant growth [5, 6]. P. ananatis strains have also been found associated with insects, including tobacco thrips that act as vectors of onion-pathogenic strains, mulberry pyralids, ticks and fleas, demonstrating its ability to persist in invertebrate hosts [7–9]. Its implication in human infections reveals its capacity for proliferation and potential to cause disease in a vertebrate host [10, 11]. The ubiquity of P. ananatis suggests that it has adapted to proliferate in a wide range of environments, and its isolation from both plant and animal hosts indicate it has adapted for cross-Kingdom colonization and pathogenesis.
The concept of the pan-genome was introduced in 2005 . The pan-genome of a bacterial species can be defined as the global gene repertoire of the species, and consists of a core genome, representing the genes present in all strains of the species, and an accessory genome composed of genes that are unique to particular strains as well as those genes that are absent from one or more of the sequenced strains [12, 13]. Core genes encode proteins that are generally involved in crucial cellular processes and are thus mostly vertically transferred from parent to progeny. Accessory genes, on the other hand, are prone to lateral gene transfer, and often encode functions related to niche adaptation [12–14]. The microbial pan-genome of a species can further be considered as ‘open’ or ‘closed’. A ‘closed’ pan-genome is highly conserved, and is typical associated with bacterial species which live in select niches, where they are secluded from the overall microbial gene pool or have a diminished capacity to acquire genes, such as Bacillus anthracis and Mycobacterium tuberculosis [13, 14]. By contrast an ‘open’ pan-genome is observed for bacterial species that can colonize and exploit several different environmental niches and can expand their accessory and pan-genome through different means of lateral gene transfer [12–14]. Four complete and four draft genomes of P. ananatis strains, isolated from various environmental sources, and with diverse lifestyles, have recently been sequenced. Here we have determined and characterized the open P. ananatis pan-genome and show its adaptive capacity to interact with hosts of both the animal and plant Kingdoms.
P. ananatisgenome statistics
General genome properties of the eight P. ananatis strains
Size (Mb) G + C% #CDS
Size (Kb) G + C% #CDS
Pan-genome statistics derived from analyses of several bacterial taxa displaying ‘open’ pan-genomes
Mean # genome CDSs
Mean # core CDSs
% core CDSs/genome
Pan-genome (# CDS) compared strains
Estimated core genome (# CDS)
Unique CDSs per additional strain
Streptococcus pneumoniae *
Haemophilus influenzae *
P. ananatishas an ‘open’ pan-genome
The pan-genome for the eight sequenced P. ananatis strains was determined by BlastP comparison of the translated CDS set, the clustering of orthologous proteins, and addition of the representatives of each orthologs cluster and strain-unique CDSs to the total pan-genome. The combined pan-genome for the eight compared P. ananatis strains encompasses 5,566 CDSs. Of these, 3,876 (69.6% of total CDSs) are core to all compared genomes (Figure 1). This implies that on average 89.5% of CDSs encoded on the genomes of each of the eight strains form part of the core genome determined for the eight compared strains. A total of 1,690 CDSs proteins (30.36% of the pan-genome total) make up the accessory fraction (Figure 1), of which an average of 108 CDSs are unique to each genome among the eight compared strains.
The P. ananatisaccessory genome encodes mainly ‘poorly characterized’ proteins
Prophage integration and integrative and conjugative elements have played a major role in the diversification of P. ananatisstrains
Integrated bacteriophage elements, or prophages, were identified in the genome sequences of the eight P. ananatis strains using Prophinder . Between two and four prophages are integrated into the replicons of each of the eight strains, and 699 accessory CDSs (41.4% of the total accessory CDSs) are encoded by these prophages, indicating that phage integration has played a substantial role in P. ananatis diversification. Between 24.2% and 74.3% of the strain-unique CDSs for each of the eight strains are localized in predicted integrated phage elements (Figure 1). This suggests that a large fraction of the strain-unique CDS complement of any new sequenced strains would likely be derived from phages. Prophages are found in two-thirds of all γ-proteobacteria and have been shown to play a major role in bacterial evolution through the horizontal transfer of genetic factors that contribute to various processes within the bacterial host, including fitness and pathogenesis . Examples of prophage-borne genes in other bacteria include those encoding a Shiga toxin in E. coli, Type III secretion system effectors in S. enterica, R- and F-type bacteriocins in Pseudomonas aeruginosa, and a superoxide dismutase (providing protection against oxidative defences within the mammalian host) in S. enterica [32–35]. The prevalence of prophage CDSs among the accessory portion of the pan-genome, suggest that they may also play a major role in the adaptive evolution of this species, potentially contributing to their ability to colonize various environmental niches and hosts.
A further 148 CDSs (8.7% of the total accessory CDSs) are encoded in integrative and conjugative elements (ICEs), which are present in the genomes of five of the sequenced strains (Figure 1). These ICEs, in other bacterial taxa, including S. enterica and Vibrio cholerae, have been shown to disseminate and confer a number of adaptive traits, including antibiotic and heavy metal resistance .
The P. ananatispan-genome encodes a number of proteins found on the genomes of bacteria associated with distinct hosts
The P. ananatispan-genome encodes proteins that may be involved in colonization of animal hosts
A total of 151 P. ananatis CDSs (2.7% of total pan-genome CDSs) encode proteins with orthologs restricted to animal-associated bacteria (AAB), including animal-pathogenic Salmonella, Escherichia and Yersinia strains, with between 61 (LMG5342) and 84 (AJ13355) AAB-specific CDSs encoded on the different P. ananatis genomes. Of these, 50 CDSs form part of the P. ananatis core genome, while 101 are found in the accessory portion of the pan-genome, suggesting possible adaptive evolution of some strains that has enabled colonization of and persistence in animal hosts. The majority of proteins encoded by the AAB-specific CDSs (110 CDSs – 66.3% of total AAB-specific CDSs) belong to the ‘poorly characterized’ super-functional category. However, a small proportion is involved in metabolism (21 CDSs) and cellular processes (17 CDSs) (Additional file 5: Table S4). Two sets of CDSs encode proteins putatively involved in the transport and metabolism of the carbohydrate substrates glucuronide and mannose, respectively. Orthologs of several P. ananatis pan-genome CDSs with a potential role in attachment, and in defense against antimicrobials are also restricted in distribution to AAB (Additional file 5: Table S4). Two CDSs unique among the sequenced P. ananatis strains to AJ13355, encode proteins with a predicted role in the biogenesis of a type 1 fimbria, with orthologs restricted to E. coli and Salmonella spp. A putative non-fimbrial autotransporter adhesin, which is common to all sequenced P. ananatis strains, shared extensive sequence identity with the AidA-I and MisL adhesins of enteropathogenic E. coli and S. enterica, respectively. These adhesins are involved in intestinal adherence in these pathogens [39, 40]. Orthologs of the S. enterica Mig-14 protein, which has been proposed to repress immune system functions , are also present in all strains. Similarly, the protein products of two P. ananatis pan-genome CDSs showed orthology to β-lactamases and their cognate transcriptional regulators in a number of AAB, including clinical strains of Enterobacter cloacae (Sfo-1/AmpR) and Citrobacter sedlakii (Sed1/SedR). These provide resistance to a broad spectrum of antibiotics used in the clinical environment [42, 43], and given their isolation from this environment [10, 11], may form the basis for antibiotic resistance of clinical P. ananatis strains.
Analysis of the P. ananatis pan-genome has thus revealed the presence of various CDSs coding for proteins with potential roles in adherence, immunity suppression, antibiotic-resistance and carbohydrate metabolism proteins that may aid in the persistence of this species in animal hosts (Additional file 5: Table S4).
The P. ananatispan-genome encodes proteins with a potential role in interactions with insect hosts
Fifteen pan-genome CDSs (0.3% of total pan-genome CDSs) share orthologs only in insect-associated bacterial genera, including Photorhabdus and Wolbachia. Between two (AJ13355 and PA13) and thirteen (LMG20103) of these IAB-specific CDSs are present in each of the individual P. ananatis genomes. In particular, one locus found in the genomes of four P. ananatis strains encodes twelve proteins showing sequence homology to a locus in Photorhabdus luminescens subsp. laumondii TTO1 and several Streptomyces spp. (Additional file 5: Table S4). Within this locus PANGEN_3511 and PANGEN_3515 encoded orthologs of two nikkomycin biosynthetic proteins in P. luminescens (plu1441 and plu1874). This antibiotic is produced by Streptomyces spp. and has acaricidal, fungicidal and insecticidal activities . PANGEN_3520 and PANGEN_3522 encoded orthologs of the Streptomyces rubellomurinus FrbC and FrbD proteins involved in production of an antimalarial compound (ABB90932-90933 - 50% average amino acid identity) . P. ananatis strains may thus have acquired a locus for the biosynthesis of a potential insecticidal peptide. As P. ananatis is frequently isolated from invertebrate hosts, the presence of such a peptide may be of interest for the biological control of insect pests [7–9].
The P. ananatispan-genome encodes proteins with a potential role in plant-microbe interactions
A large number of the P. ananatis pan-genome CDSs (1,249 CDSs – 22.4% of total pan-genome content) shared orthology with CDSs restricted to plant-associated bacteria, and between 933 (PA4) and 985 (LMG2665T) of these are encoded on each individual P. ananatis genome. This finding concurs with the frequent isolation of P. ananatis from the plant environment . Of these CDSs, 849 formed part of the pan-genome core, while 400 were associated with the accessory genome. While the majority of PAB-specific CDSs were found in common with both enterobacterial and non-enterobacterial plant-associated species, a relatively large number (200 CDSs – 16% of total PAB-specific CDSs) shared orthology only with CDSs restricted to non-enterobacterial PAB. This suggests that extensive horizontal exchange has occurred between P. ananatis and non-enterobacterial PAB. A total of 200 PAB-specific CDSs are localized on the pPANA1 pan-plasmid (50.5% of the total pan-plasmid CDSs). Previously we showed that the pPANA1 plasmid is part of the Large Pantoea Plasmid group (LPP-1) common to all sequenced Pantoea spp. The LPP-1 plasmids share a small set of common core CDSs and a much larger accessory component, and we postulated that they play a major role in the ecological diversification of the genus [46, 47]. The large number of PAB-specific CDSs (Figure 4) indicates that the pPANA1 plasmid likely plays a major role in the adaptation of P. ananatis to colonize and interact with plants.
As is the case for AAB-specific CDSs, the largest proportion of PAB-specific CDSs encoded proteins that belong to the ‘poorly characterized’ super-functional category (46.2% of the total PAB-specific CDSs), but a substantial number of PAB-specific CDSs encode proteins involved in metabolism (30.4%) and other cellular processes (11.5%). These may play a role in efficient colonization, nutrient utilization and persistence in or on the plant and/or in other plant-microbe interactions. An extensive set of PAB-specific CDSs encode proteins with a role in the transport and metabolism of carbohydrates (Additional file 6: Table S5), which may facilitate the uptake and metabolism of plant-derived carbohydrates. A number of orthologs of proteins involved in the degradation of plant carbohydrates are also encoded by PAB-specific CDSs present in all P. ananatis strains, including a predicted endo-1,4-β-xylanase, two polygalacturonases, a putative pectin acetylesterase, as well as a predicted cellulase with extensive sequence identity to the minor cellulose Cel8Y of Dickeya dadantii  (Additional file 6: Table S5). Several PAB-restricted amino acid transport and metabolism systems are also encoded on the P. ananatis pan-genome, including orthologs of the opine octopine, released from plant tumors induced by Agrobacterium tumefaciens  and the Amadori compound deoxyfructosyl glutamine, found in rotting fruits and vegetables, and in tumors caused by chrysopine-type Agrobacterium strains  (Additional file 6: Table S5). These compounds could furthermore serve as carbon, nitrogen and energy sources for P. ananatis in the plant. Several PAB-specific proteins with a predicted role in iron uptake and metabolism were also found to be encoded on the P. ananatis pan-genome. These included a predicted hydroxamate siderophore transporter, siderophore receptor, ferric dicitrate sensor components FecI/FecR and a PAB-specific TonB-ExbBD complex for outer membrane iron transport (Additional file 6: Table S5). These PAB-specific proteins may allow P. ananatis to actively contest for the limited iron available in the plant environment .
Several CDSs encoding PAB-restricted proteins potentially involved in protection against plant defenses are present in the P. ananatis pan-genome. PANGEN_04534-4535 encode orthologs of the protein Ohr and its transcriptional regulator OhrR, which are involved in resistance to organic hydroperoxides produced by plants in defense to pathogen infection . Several PAB-restricted multidrug efflux pumps that could play a role in the extrusion of plant-produced antimicrobials and β-lactamases, which may specifically degrade plant antimicrobials, are encoded on the P. ananatis pan-genome (Additional file 6: Table S5). PANGEN_05563 encodes an ortholog of the cyclic β-1,2-glucan polysaccharide biosynthetic protein NdvB. This polysaccharide has been shown to provide protection to Xanthomonas campestris against localized and systemic defenses in the host plant . A PAB-specific locus on the pPANA1 plasmid of all eight P. ananatis strains encodes orthologs of the proteins BudABCR, which are involved in the production of 2,3-butanediol. This volatile has been shown to promote plant growth and induce systemic resistance in the plant host [54, 55], which may be linked to the biological role as plant growth promoter ascribed to strains of this species [5, 6].
The P. ananatispan-genome encodes proteins with potential roles in plant- and animal-pathogenesis
As strains of P. ananatis have been found to be pathogenic on a broad range of plant hosts as well as humans, the pan-genome was analyzed to identify potential molecular determinants underlying its pathogenicity. Our analyses revealed the absence of many of the factors that are central to the pathogenicity and virulence arsenal of related plant and animal pathogens, including animal toxins, hemolysins, phytotoxins, several secretion systems (Type II, III and IV) and their associated effectors. However, several genes and loci with orthology to characterized pathogenicity determinants in related animal and plant pathogens could be identified in the P. ananatis pan-genome.
Analysis of the PAB-specific complement also showed the presence of a CDS encoding an ice-nucleation protein with orthologs restricted to plant-pathogenic strains of P. ananatis, Pantoea agglomerans, X. campestris and P.s syringae. This protein induces wounds in frost-damaged plants and is postulated to allow these pathogens to gain access to host tissues . A locus on the pPANA1 plasmids of P. ananatis LMG2665T and B1-9 encodes proteins (PANGEN_05466-5474) with orthology to proteins for the biosynthesis of non-ribosomal peptide synthases/polyketide synthases (NRPS/PKS). The potential function of this locus in NRP/PK biosynthesis was further substantiated by comparison of the locus against the antiSMASH server . Many phytopathogenic bacteria have been shown to carry CDSs encoding NRPS/PKS that are required for the production of phytotoxins, e.g. P. syringae syringomycin and syringopeptin, and P. atrosepticum coronofacic acid conjugates . Orthologs of the proteins encoded in the P. ananatis NRPS/PKS locus were found to be restricted to PAB (E. amylovora ATCC BAA-2158 - EAIL5_2884-2892; 72% average amino acid identity) and may thus potentially encode synthases for the production of a phytotoxin. However, the biological role of the NRPS/PKS products in P. ananatis will need to be determined.
Pantoea ananatis is ubiquitous in the environment and has an inherent capacity to survive, proliferate and form intimate relationships with plants, as well as insect and human hosts . In particular, its frequent isolation from both plant and animal hosts suggests it has adapted to colonize, proliferate and potentially cause disease in these hosts. Here we analyzed the genome sequences of eight P. ananatis strains. As has been observed for other members of the Enterobacteriaceae, P. ananatis exhibits an ‘open’ pan-genome, which is mainly influenced by the integration of phage elements, but also by integrative conjugative elements and other insertion elements. Phages play a significant role in bacterial evolution, transferring fitness and pathogenicity factors to their bacterial host . They could therefore represent major adaptive drivers for P. ananatis, allowing strains of this species to colonize and interact with both plant and animal hosts.
Analysis of the P. ananatis pan-genome CDS complement revealed the presence of a large number of proteins restricted in distribution to plant- and animal-associated bacteria (PAB and AAB). These include a number of factors that could serve as putative tools for P. ananatis adherence and colonization of host tissues, to utilize nutrients and persist within a host(s), and potentially cause disease. However, it cannot be excluded that common mechanisms underlying colonization, persistence and pathogenicity exist among bacteria that are associated with both plant and animal hosts. The ability of a bacterium to make a cross-Kingdom jump is dependent on several prerequisites, including the close and frequent contact with the novel host, the ability to overcome host defences, and the capacity for horizontal acquisition of genes encoding factors that enable the bacterium to persist in its new host . The frequent isolation of P. ananatis from various different environments and the (pan-)genomic evidence of a bacterial species well-adapted towards survival in and interaction with different hosts, provide a primary indication of the ecological success of P. ananatis and how it may have evolved to interact with cross-Kingdom hosts.
Genome comparisons and construction of the P. ananatispan-genome
The genome sequences from eight P. ananatis strains, four complete and four partially assembled genomes (Table 1), were included in the study. The partial genomes were annotated using FgenesB . CDS sets were standardized by local BlastN analysis to identify ORFs which may not have been predicted for a particular genome. The combined nucleotide sequences of the chromosome and pPANA1 plasmid, for the P. ananatis strains for which complete genomes were available, were aligned using Mauve v. 2.3.1. . The translated CDS sets for each of the eight genomes were pair-wise compared by local BlastP analysis using Bioedit 7.1.11 software package . The comparison was performed using the reciprocal best hit approach (RBBH), whereby orthologs were assumed when a Blast hit of a query protein in the compared subject protein set also returned the query protein sequence as the best Blast hit when it in turn was compared by BlastP analysis against the query strain protein set . The number of orthologous CDSs and average amino acid identities (sum of the amino acid identities for each compared protein divided by the aligned length of the protein) for each combination of pair-wise compared proteins sets were determined. Orthologs were defined using cut-off values (>70% amino acid identity and >70% sequence coverage for the query and hit) . Using these orthology parameters and localized BlastP comparison, the orthologous CDSs from each of the genomes were clustered , where each cluster represented the set of RBBH orthologs for each distinct CDS across all the compared genomes. A representative of each cluster, the longest member sequence of each cluster for CDSs shared by one or more strain, as well as CDSs unique to specific P. ananatis strains, were incorporated in a single pan-genome file. The translated protein products of the entire pan-genome CDS set were compared against the Conserved Orthologous Groups database using COGnitor  to determine the COG functional and super-functional category to which they belong. Prophages and phage proteins were identified by searching against the ACLAME database using Prophinder . A diagram of the eight CDS sets compared against the pan-genome CDS set was constructed on the basis of the above localized BlastP analysis results using GenomeDiagram .
The size of the pan-genome for the eight sequenced strains, as well as the core and accessory fractions were determined by localized BlastP analysis with the translated CDS sets of each of the sequence P. ananatis strain against the pan-genome CDS set. The core genome, signifying all pan-genome CDSs common to all eight strains, and the accessory genome, incorporating those CDSs which are absent in or more of the strains or were unique to the genome of a particular strain were tabulated, and the pan-genome determined as the sum of the core and accessory CDSs.
The sequential inclusion of the CDS sets of each of the eight genomes in all possible combinations was used to determine the core CDSs, accessory CDSs shared by more than one but not all strains, and those unique to a single strain, as a function of the number of genomes (n) in the comparison (where n = 1,2,…8). The estimated strain-specific CDSs and core CDSs for the species, beyond the scope of eight sequenced strains if the genomes of additional strains were sequenced (i.e. n→ ∞), were extrapolated by fitting the data for the n = 1→8 comparison combinations above to an exponential decay functions as per . The data was fitted to the function using the generalized least squares (gnls) algorithm of the nlme (linear and non-linear mixed-effects models) package in R . The estimated data points for n→∞ obtained from the function along with the actual data points from the comparison of the eight P. ananatis CDS sets were plotted in graphs (Figure 2A and B) of the number of genomes versus the number of core/strain-specific CDSs. In order to extrapolate the pan-genome size for the species, the strain-unique CDSs and average number of CDSs per compared genome (for n = 2→8 genomes compared) were incorporated into an algebraic formula as per .
Comparison between the P. ananatispan-genome and all available genomes
The CDSs encoded by P. ananatis were compared by BlastP analysis against the NCBI non-redundant (nr) protein database . For this comparison, orthology was assumed for proteins sharing >30% amino acid identity and 70% sequence coverage between the query and hit . On the basis of the Blast hits, CDSs were identified that shared orthologs in bacteria occupying distinct ecological niches, namely those associated with animals (AAB), insects (IAB) and plants (PAB), as determined from available information of their source of isolation. For the purpose of this grouping, only those bacteria that are specifically associated with animals and plant tissues and/or the rhizosphere environment were taken into consideration, while members that are frequently found associated with hosts of both Kingdoms, such as Klebsiella and Enterobacter sp., were disregarded.
A diagram of the comparison between the translated protein products encoded by the P. ananatis pan-genome CDS set and the protein sets of 54 members of the Enterobacteriaceae, encompassing all the genera for which complete genomes were available (Additional file 7: Table S6), was constructed using GenomeDiagram , using the RBBH approach for localized BlastP comparison. Orthologs were considered when proteins shared >50% amino acid identity and >70% coverage between the hit and query sequences.
Availability of supporting data
The complete genome sequences of P. ananatis LMG20103 (chromosome + plasmid: NC_013956.2), AJ13355 (chromosome: NC_017531.1; plasmid: NC_017533.1), PA13 (chromosome: NC_017554.1; plasmid: NC_017553.1) and LMG5342 (chromosome: NC_016816.1; plasmid: NC_016817.1), as well as the draft genomes of P. ananatis B1-9 (CAEI00000000 and CAEJ00000000), LMG2665T ((JMJJ00000000), PA4 (JMJK00000000) and BD442 (JMJL00000000) are publically available on the NCBI database under the given accession numbers. The protein datasets for all eight P. ananatis strains, as well as the R scripts, input and output files for the pan-genome calculations and pan-genome graphs are available in the LabArchives repository .
This study was partially supported by the University of Pretoria Postdoctoral Fellowship Program, National Research Foundation (NRF), the Tree Protection Co-operative Programme (TPCP), the NRF/Dept. of Science and Technology Centre of Excellence in Tree Health Biotechnology (CTHB), and the THRIP support program of the Department of Trade and Industry, South Africa. IKT and PRJB were supported by a grant from the Scottish Government’s Rural and Environmental Science and Analytical Services (RESAS) division.
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