Forestry and Agricultural Biotechnology Institute

Colletotrichum destructivum is an important plant pathogen, mainly of forage and grain legumes including clover, alfalfa, cowpea and lentil, but has also been reported as an anthracnose pathogen of many other plants worldwide. Several Colletotrichum isolates, previously reported as closely related to C. destructivum, are known to establish hemibiotrophic infections in different hosts. The inconsistent application of names to those isolates based on outdated species concepts has caused much taxonomic confusion, particularly in the plant pathology literature. A multilocus DNA sequence analysis (ITS, GAPDH, CHS-1, HIS3, ACT, TUB2) of 83 isolates of C. destructivum and related species revealed 16 clades that are recognised as separate species in the C. destructivum complex, which includes C. destructivum, C. fuscum, C. higginsianum, C. lini and C. tabacum. Each of these species is lecto-, epi- or neotypified in this study. Additionally, eight species, namely C. americae-borealis, C. antirrhinicola, C. bryoniicola, C. lentis, C. ocimi, C. pisicola, C. utrechtense and C. vignae are newly described.

Herein the members of the Subcommittee on Taxonomy of Rhizobia and Agrobacteria of the International Committee on Systematics of Prokaryotes review recent developments in rhizobial and agrobacterial taxonomy and propose updated minimal standards for the description of new species (and genera) in these groups. The essential requirements (minimal standards) for description of a new species are (1) a genome sequence of at least the proposed type strain and (2) evidence for differentiation from other species based on genome sequence comparisons. It is also recommended that (3) genetic variation within the species is documented with sequence data from several clearly different strains and (4) phenotypic features are described, and their variation documented with data from a relevant set of representative strains. Furthermore, it is encouraged that information is provided on (5) nodulation or pathogenicity phenotypes, as appropriate, with relevant gene sequences. These guidelines supplement the current rules of general bacterial taxonomy, which require (6) a name that conforms to the International Code of Nomenclature of Prokaryotes, (7) validation of the name by publication either directly in the International Journal of Systematic and Evolutionary Microbiology or in a validation list when published elsewhere, and (8) deposition of the type strain in two international culture collections in separate countries.

BACKGROUND: Terpenoids are abundant in the foliage of Eucalyptus, providing the characteristic smell as well as being valuable economically and influencing ecological interactions. Quantitative and qualitative inter- and intra- specific variation of terpenes is common in eucalypts. RESULTS: The genome sequences of Eucalyptus grandis and E. globulus were mined for terpene synthase genes (TPS) and compared to other plant species. We investigated the relative expression of TPS in seven plant tissues and functionally characterized five TPS genes from E. grandis. Compared to other sequenced plant genomes, Eucalyptus grandis has the largest number of putative functional TPS genes of any sequenced plant. We discovered 113 and 106 putative functional TPS genes in E. grandis and E. globulus, respectively. All but one TPS from E. grandis were expressed in at least one of seven plant tissues examined. Genomic clusters of up to 20 genes were identified. Many TPS are expressed in tissues other than leaves which invites a re-evaluation of the function of terpenes in Eucalyptus. CONCLUSIONS: Our data indicate that terpenes in Eucalyptus may play a wider role in biotic and abiotic interactions than previously thought. Tissue specific expression is common and the possibility of stress induction needs further investigation. Phylogenetic comparison of the two investigated Eucalyptus species gives insight about recent evolution of different clades within the TPS gene family. While the majority of TPS genes occur in orthologous pairs some clades show evidence of recent gene duplication, as well as loss of function.

The present paper represents the second contribution in the Genera of Fungi series, linking type species of fungal genera to their morphology and DNA sequence data, and where possible, ecology. This paper focuses on 12 genera of microfungi, 11 of which the type species are neo- or epitypified here: Allantophomopsis (A. cytisporea, Phacidiaceae, Phacidiales, Leotiomycetes), Latorua gen. nov. (Latorua caligans, Latoruaceae, Pleosporales, Dothideomycetes), Macrodiplodiopsis (M. desmazieri, Macrodiplodiopsidaceae, Pleosporales, Dothideomycetes), Macrohilum (M. eucalypti, Macrohilaceae, Diaporthales, Sordariomycetes), Milospium (M. graphideorum, incertae sedis, Pezizomycotina), Protostegia (P. eucleae, Mycosphaerellaceae, Capnodiales, Dothideomycetes), Pyricularia (P. grisea, Pyriculariaceae, Magnaporthales, Sordariomycetes), Robillarda (R. sessilis, Robillardaceae, Xylariales, Sordariomycetes), Rutola (R. graminis, incertae sedis, Pleosporales, Dothideomycetes), Septoriella (S. phragmitis, Phaeosphaeriaceae, Pleosporales, Dothideomycetes), Torula (T. herbarum, Torulaceae, Pleosporales, Dothideomycetes) and Wojnowicia (syn. of Septoriella, S. hirta, Phaeosphaeriaceae, Pleosporales, Dothideomycetes). Novel species include Latorua grootfonteinensis, Robillarda africana, R. roystoneae, R. terrae, Torula ficus, T. hollandica, and T. masonii spp. nov., and three new families: Macrodiplodiopsisceae, Macrohilaceae, and Robillardaceae. Authors interested in contributing accounts of individual genera to larger multi-authored papers to be published in IMA Fungus, should contact the associate editors listed for the major groups of fungi on the List of Protected Generic Names for Fungi (www.generaoffungi.org).

Higher plants contain a family of cellulose synthase catalytic subunit (CesA) genes that encode components of an enzyme complex embedded in the cell membrane. Recent studies in several higher plant species have demonstrated that two groups of CesA genes exist, associated with either primary or secondary cell wall deposition. We cloned six full-length CesA cDNAs from Eucalyptus grandis W. Hill ex Maiden (EgCesA1 through 6) and determined their expression patterns in a variety of organs from an adult tree. The six EgCesA genes encode predicted proteins of 978 to 1097 amino acid residues, each of which contains all of the key regions and motifs characteristic of functional CESA proteins. The predicted proteins share limited amino acid identity with each other, ranging from 61 to 70%. In contrast, similar CESA proteins from higher plant species exhibit 81 to 90% identity with the six EgCESAs. Gene expression analysis using quantitative reverse-transcription polymerase chain reaction indicated that transcripts of EgCesA1 to 3 were abundant in tissues enriched with cells laying down secondary cell walls (e.g., xylem), but were weakly expressed in tissues undergoing primary growth (e.g., unfolding leaves). Expression of EgCesA4 and EgCesA5 was upregulated in tissues rich in rapidly dividing cells undergoing primary wall synthesis, whereas EgCesA6 was weakly expressed in all tissues analyzed. These results suggest that Eucalyptus, like other higher plants, expresses two contrasting groups of apparently co-regulated CesAs involved in either primary or secondary cell wall biosynthesis.

Sphaeropsis sapinea is a fungal endophyte of Pinus spp. that can cause disease following predisposition of trees by biotic or abiotic stresses. Four morphotypes of S. sapinea have been described from within the natural range of the fungus, while only one morphotype has been identified on exotic pines in the Southern Hemisphere. The aim of this study was to develop robust polymorphic markers that could be used in both taxonomic and population studies. Inter-short-sequence-repeat primers containing microsatellite sequences and degenerate anchors at the 5' end were used to target microsatellite-rich areas in an S. sapinea isolate. PCR amplification using an annealing temperature of 49 degrees C resulted in profiles containing 5 to 10 bands. These bands were cloned and sequenced, and new short-sequence-repeat (SSR) primer pairs were designed that flanked microsatellite-rich regions. Eleven polymorphic SSR markers were tested on 40 isolates of S. sapinea representing different morphotypes as well as on 2 isolates of the closely related species Botryosphaeria obtusa. The putative I morphotype was found to be identical to B. obtusa. Otherwise, the markers clearly distinguished the remaining three morphotypes and, furthermore, showed that the C morphotype was more closely related to the A than the B morphotype. The B morphotype was the most genetically diverse, and the isolates could be further divided based on their geographic origins. Sequencing of different alleles from each locus showed that the most polymorphic markers had mutations within a microsatellite sequence.

The International Society of Plant Pathology Committee on the Taxonomy of Plant Pathogenic Bacteria has responsibility to evaluate the names of newly proposed pathovars for adherence to the International Standards for Naming Pathovars of Phytopathogenic Bacteria. Currently, the Comprehensive List of Names and the List of New Names of Plant Pathogenic Bacteria provide the authoritative register of names of bacterial plant pathogens. In this manuscript we up-date the list of names by cataloguing and evaluating names of plant pathogenic bacteria published in 2011 and 2012. We provide those names that have been validly and effectively published in this time frame, the proposed names that we judged to be invalid, and names published earlier that did not make the previous lists.

Chrysoporthe canker is one of the most important diseases of plantation-grown Eucalyptus spp. in tropical and subtropical regions worldwide. For many years, the disease was reported to be caused by the fungal pathogen Cryphonectria cubensis. Recent DNA-based studies have shown that the fungus in South Africa is not conspecific with Chr. cubensis and it was recently described in the new genus Chrysoporthe as Chrysoporthe austroafricana. Chr. austroafricana is known only from South Africa, where it causes severe cankers on Eucalyptus spp. and on ornamental Tibouchina trees, both of which have been introduced into South Africa. The origin of Chr. austroafricana is unknown, but it is possible that it expanded its host range from native trees related to Eucalyptus and Tibouchina spp. to these exotic hosts. Subsequent surveys of some indigenous South African Myrtales led to the discovery of fruiting structures resembling those of Chr. austroafricana on native Syzygium cordatum and S. guineense. The fungus from these Syzygium spp. was identified as Chr. austroafricana based on morphological characteristics and β-tubulin gene sequences. Pathogenicity trials showed that Chr. austroafricana is more virulent on exotic Eucalyptus spp. than on native S. cordatum. This study represents the first report of Chr. austroafricana from native hosts in South Africa and adds credence to the view that the fungus could be native to this country.

The eight spined larch bark beetles infest various species of Larix in Europe and Asia. Ips cembrae is the only Ips species with larch as its main host. Ips subelongatus, Ips fallax, Ips shinanonensis and Ips cembrae var. engadinensis are treated as synonyms of I. cembrae. These three putative species and the one variety are distinguished by their host tree and geographic distribution, as it is not possible to distinguish them on the basis of morphological differences. Beetles were collected from European and Asian populations, and from hosts and geographic areas where the species were first found and described and in their natural ranges of distribution. These beetles were used to study the phylogenetic relationships of the eight spined larch bark beetles. A region of the mitochondrial gene was analysed and the blue-stain fungi associated with I. cembrae in Europe were investigated and compared with those recorded as associated with the larch bark beetle in Japan. Only minor sequence differences were detected between the populations in Europe and Asia. However, the European populations differed by 4.3% from the Asian populations. The phylogenetic analysis placed the European and Asian haplotypes in significantly distinct clusters. This distinction was supported by the finding of an insertion/deletion in a non-coding region of the mitochondrial DNA. Furthermore, there are differences in the fungi associated with the eight spined larch bark beetles in Europe and Japan. The results suggest that the I. cembrae complex contains at least two taxa: I. cembrae infesting larch in Europe and I. subelongatus infesting larch in Asia.

Species of the ascomycete family Botryosphaeriaceae are important pathogens of trees and other woody plants. This chapter focuses on the epidemiology and management of cankers and other diseases caused by the Botryosphaeriaceae. Information is given on their detection, infection biology, insect associations, as well as management strategies and tactics, which include avoidance, exclusion, eradication, protection by using fungicides, host resistance, and integrated method of control.

Calonectria (Ca.) species and their Cylindrocladium (Cy.) anamorphs are well-known pathogens of forest nursery plants in subtropical and tropical areas of the world. An investigation of the mortality of rooted Pinus cuttings in a commercial forest nursery in Colombia led to the isolation of two Cylindrocladium anamorphs of Calonectria species. The aim of this study was to identify these species using DNA sequence data and morphological comparisons. Two species were identified, namely one undescribed species, and Cy. gracile, which is allocated to Calonectria as Ca. brassicae. The new species, Ca. brachiatica, resides in the Ca. brassicae species complex. Pathogenicity tests with Ca. brachiatica and Ca. brassicae showed that both are able to cause disease on Pinus maximinoi and P. tecunumanii. An emended key is provided to distinguish between Calonectria species with clavate vesicles and 1-septate macroconidia.

Ophiostoma ips is a common fungal associate of various conifer-infesting bark beetles in their native ranges and has been introduced into non-native pine plantations in the Southern Hemisphere. In this study, we used 10 microsatellite markers to investigate the population biology of O. ips in native (Cuba, France, Morocco and USA) and non-native (Australia, Chile and South Africa) areas to characterize host specificity, reproductive behaviour, and the potential origin as well as patterns of spread of the fungus and its insect vectors. The markers resolved a total of 41 alleles and 75 haplotypes. Higher genetic diversity was found in the native populations than in the introduced populations. Based on the origin of the insect vectors, the populations of O. ips in Australia would be expected to reflect a North American origin, and those in Chile and South Africa to reflect a European origin. However, most alleles observed in the native European population were also found in the native North American population; only the allele frequencies among the populations varied. This admixture made it impossible to confirm the origin of the introduced Southern Hemisphere (SH) populations of O. ips. There was also no evidence for specificity of the fungus to particular bark beetle vectors or hosts. Although O. ips is thought to be mainly self-fertilizing, evidence for recombination was found in the four native populations surveyed. The higher genetic diversity in the North American than in the European population suggests that North America could be the possible source region of O. ips.

Losses in the production of avocado (Persea americana (Mill.)) are incurred due to Phytophthora root rot (PRR), a disease of the feeder roots that results in tree-dieback and eventual tree death. Avocado is also a flood-sensitive species and flooding exacerbates the effects of PRR. The avocado industry relies on the use of rootstocks tolerant to PRR to minimise losses. The present study compared the gas exchange and chlorophyll fluorescence responses of avocado rootstock plants of ‘Dusa™’, the current South African industry standard, with ‘Duke 7’, and the selections R0.12 and R0.06 which show reduced and superior tolerance to PRR, respectively. A decline in stomatal conductance (gs) and net CO2 assimilation (PN) over the 30 day evaluation period were early responses to flooding. ‘Dusa™’, the more tolerant rootstock plants, demonstrated a better recovery in PN and gs in response to inoculation; however, both rootstocks performed poorly under flooded conditions. A decline in PN in infected ‘Duke 7’ plants appeared to be associated with stomatal limitations due to reduced stomatal conductance. The decline in PN and gs was not apparent in infected ‘Dusa™’ plants. Non-stomatal limitations to PN in rootstock plants exposed to flooding were also evident as indicated by increases in the ratio of internal to atmospheric CO2 concentrations (Ci/Ca). Impaired photosynthetic capacity in flooded rootstock plants was reflected by reduced photosystem II efficiency and photochemical quenching. In comparison to ‘Dusa™’, R0.12 rootstock plants showed reduced PN and gs following inoculation with Phytophthora cinnamomi whereas the more tolerant R0.06 rootstock plants revealed sustained photosynthetic activity. Interestingly R0.06 was the only rootstock able to maintain PN and gs in non-inoculated, flooded plants.

Armillaria root rot symptoms were observed on native Protea and Leucadendron (Proteaceae) species in Kirstenbosch Botanical Gardens in the Western Cape Province of South Africa. Intergenic spacer (IGS)‐1 polymerase chain reaction (PCR) restriction fragment‐length polymorphism (RFLP) profiling indicated the presence of at least two Armillaria species. The profiles of two isolates were identical to those of A. mellea s. str., originating in Europe. Phylogenetic analyses incorporating internal transcribed spacer (ITS) and IGS‐1 sequence data identified the remaining isolates as closely related to A. calvescens , A. gallica , A. jezoensis and A. sinapina . These isolates displayed mating compatibility with A. gallica. From the RFLP profiles, sequencing results and sexual compatibility studies, it is concluded that the two species on Proteaceae in Kirstenbosch represent A. mellea and A. gallica . These are northern hemisphere fungi that have apparently been accidentally introduced into South Africa. This is the second report of Armillaria being introduced into South Africa. The introduction probably occurred early in the colonization of Cape Town, when potted plants from Europe were used to establish gardens.

Phytophthora cinnamomi causes Phytophthora root rot (PRR) in avocado (Persea americana), an important disease that causes severe economic losses to the avocado industry globally. To date, no PRR-resistant avocado rootstock variety has been discovered, although certain rootstock varieties have been shown to be more tolerant than others. In this study, we developed an accurate, low cost assay for in planta quantification of P. cinnamomi to evaluate disease tolerance. A nested real-time polymerase chain reaction assay was developed to sensitively detect pathogen DNA in plant tissues. Root samples from a highly tolerant (Dusa) and less tolerant (R0.12) rootstock were collected at 0, 3, 7, 14, and 21 days after inoculation with P. cinnamomi and used for pathogen quantification. Nested primers developed in this study were specific and sensitive and could detect P. cinnamomi in root tissues. The amount of P. cinnamomi quantified in roots was significantly higher in the less-tolerant R0.12 plants when compared with the highly tolerant Dusa plants at all time points. This study has confirmed the known status of disease tolerance of Dusa and R0.12 avocado rootstocks in a quantitative manner and provides a reliable molecular tool to assist with industry breeding programs for the selection of PRR-resistant avocado rootstock varieties.

Despite the availability of high-throughput transcript profiling technology, little is known about tissue-specific gene expression patterns in the wood-forming tissues of Eucalyptus plantation tree species. We used cDNA-amplified fragment length polymorphism (AFLP) analysis in combination with infrared fragment detection and semi-automated band quantification to profile gene expression in a 6-year-old, fast- growing Eucalyptus tree. The expression profiles of 6385 transcript-derived fragments (TDFs) were analyzed across four major woody tissues (mature xylem, immature xylem, phloem and cork) collected from two stem positions, to provide a global view of transcript abundance and variability in the Eucalyptus stem. About 21% of the TDFs were differentially expressed and could be grouped into clusters representing co- expressed genes. A total of 71 TDFs representing different gene clusters were isolated and characterized. These included genes implicated in cell fate, signal transduction and cell wall biosynthesis, processes closely associated with xylogenesis. Analysis of the expression levels of selected TDFs by quantitative RT-PCR corroborated the TDF quantification and confirmed that cDNA-AFLP analysis is a highly efficient and accurate tool for transcript profiling and gene discovery in wood-forming tissues of tree species.

At present, much attention is being given to the potential of plant pathogens, including plant-pathogenic bacteria, as biological weapons/bioterror weapons. These two terms are sometimes used interchangeably and there is need for care in their application. It has been claimed that clandestine introduction of certain plant-pathogenic bacteria could cause such crop losses as to impact so significantly on a national economy and thus constitute a threat to national security. As a separate outcome, it is suggested that they could cause serious public alarm, perhaps constituting a source of terror. Legislation is now in place to regulate selected plant-pathogenic bacteria as potential weapons. However, we consider it highly doubtful that any plant-pathogenic bacterium has the requisite capabilities to justify such a classification. Even if they were so capable, the differentiation of pathogens into a special category with regulations that are even more restrictive than those currently applied in quarantine legislation of most jurisdictions offers no obvious benefit. Moreover, we believe that such regulations are disadvantageous insofar as they limit research on precisely those pathogens most in need of study. Whereas some human and animal pathogens may have potential as biological or bioterror weapons, we conclude that it is unlikely that any plant-pathogenic bacterium realistically falls into this category.

The purpose of this study was to evaluate the resistance to coffee leaf rust (CLR) caused by Hemileia vastatrix and to Ceratocystis canker (Cc) in coffee genotypes derived from crosses of Coffea arabica var. Caturra with accessions of C. canephora backcrossed to Caturra. Twenty-three F3BC1 progenies including C. arabica var. Caturra and var. Colombia as controls were established in a field experiment. CLR evaluations were made during five years of natural infection, using an incidence rating scale. For Cc, artificial stem inoculations were made with an isolate of Ceratocystis colombiana and the results were assessed after one year. The selection process also included agronomic aspects such as plant height, canopy diameter, number of branch pairs, yield and grain characteristics. Twenty progenies showed >70% of rust resistance. Twelve progenies exhibited >80% of Cc resistance, while no resistance was observed in either of the controls. Only three progenies performed well for all criteria, including resistance to both pathogens and agronomic characteristics.

Endothia gyrosa is a canker pathogen best known as the causal agent of pin oak blight in North America, and causes cankers on other woody hosts such as Castanea spp. and Liquidambar spp. In South Africa, Australia and Tasmania, a fungus identified as E. gyrosa has been recorded on Eucalyptus spp. Some morphological differences exist between the North American fungus and the isolates from Eucalyptus . Phylogenetic relationships between E. gyrosa from North America and E. gyrosa from South Africa and Australia, as well as that of the related fungi Cryphonectria parasitica and C. cubensis , were studied using PCR‐based restriction fragment length polymorphism (RFLP) and sequences of the internal transcribed spacer (ITS) region of the rRNA operon. Endothia gyrosa isolates from South Africa produced the same RFLP banding patterns as those from Australia, which differed markedly from North American isolates of E. gyrosa . In a phylogram based on the DNA sequences, the Australian and South African isolates of E. gyrosa resided in a single, well resolved clade, distinct from North American isolates. Isolates of C. parasitica grouped in the same clade as the South African and Australian isolates of E. gyrosa , but C. cubensis was distantly related to them. The molecular data suggest that the E. gyrosa isolates from South Africa and Australia represent a distinct taxon, and probably belong to the genus Cryphonectria .

After recent changes to the taxonomy of the Botryosphaeriaceae species with diplodia-like (= dark, ovoid, often pigmented) conidia are considered to belong to at least three genera including Diplodia, Lasiodiplodia and Dothiorella. In a recent molecular phylogenetic study it became apparent that two groups of isolates with diplodia-like conidia required taxonomic revision. One group of isolates originated from Cupressus sempervirens in Greece and Cyprus and had been identified as D. pinea f. sp. cupressi based on morphological characteristics. The other isolates originated from a Casuarina sp. in Australia and were superficially similar to those in the first group based on their morphologically similar diplodia-like conidia. The aim of this study was to resolve the taxonomy of these two groups of isolates by combining the information from the multiple gene genealogies with morphological characters. The results showed that the isolates from C. sempervirens in Greece and Cyprus represent D. cupressi. The isolates from Casuarina in Australia belong to the more distantly related genus Dothiorella and represent a distinct species that is described here as Do. casuarini sp. nov.