A serious canker disease of Eucalyptus in South Africa caused by a new species of Coniothyrium

Eucalyptus spp. are being propagated extensively as exotics in plantations in South Africa, and many other parts of the world. In South Africa, a number of diseases result in serious losses to this resource. This paper describes a new and very damaging stem canker disease, which has recently appeared on plantation-grown eucalyptus in South Africa. The disease, first noted in an isolated location in Zululand is now common in other parts of the country, and is typified by discrete necrotic lesions on stems. These lesions coalesce to form large, gum-impregnated cankers and malformed stems. The causal agent of the disease, as inferred from pathogenicity tests, is a new species of Coniothyrium described here as C. zuluense. This fungus is a serious impediment to eucalypt propagation in South Africa, and is most likely a threat to similar forest industries elsewhere in the world.     

First record of Colletogloeopsis zuluense comb. nov., causing a stem canker of Eucalyptus in China

Coniothyrium zuluense causes a serious canker disease of Eucalyptus in various parts of the world. Very little is known regarding the taxonomy of this asexual fungus, which was provided with a name based solely on morphological characteristics. In this study we consider the phylogenetic position of C. zuluense using DNA-based techniques. Distance analysis using 18S and ITS regions revealed extensive sequence divergence relative to the type species of Coniothyrium, C. palmarum and species of Paraconiothyrium. Coniothyrium zuluense was shown to be an anamorph species of Mycosphaerella, a genus that includes a wide range of Eucalyptus leaf and stem pathogens. Within Mycosphaerella it clustered with taxa having pigmented, verruculose, aseptate conidia that proliferate percurrently and sympodially from pigmented conidiogenous cells arranged in conidiomata that vary from being pycnidial to acervular. The genus Colletogloeopsis is emended to include species with pycnidial conidiomata, and the new combination Colletogloeopsis zuluense is proposed. This is also the first report of the pathogen from China where it is associated with stem cankers on Eucalyptus urophylla.     

Development of polymorphic microsatellite markers for the fungal tree pathogen Cryphonectria eucalypti

Polymorphic microsatellite DNA markers were developed from a single spore isolate of Cryphonectria eucalypti collected from a Eucalyptus stem canker in South Africa. Markers were obtained using the enrichment technique known as fast isolation by AFLPs of sequences containing repeats (FIASCO). Ten polymorphic markers were isolated, of which, two were discarded due to their high polymorphism in the flanking region. The mean number of alleles produced by the remaining eight markers from 20 isolates was 7.25, and alleles per locus ranged from four to 12. The markers will be used to study populations of C. eucalypti.     

Novel hosts of the Eucalyptus canker pathogen Chrysoporthe cubensis and a new Chrysoporthe species from Colombia

The pathogen Chrysoporthe cubensis (formerly Cryphonectria cubensis) is best known for the important canker disease that it causes on Eucalyptus species. This fungus is also a pathogen of Syzygium aromaticum (clove), which is native to Indonesia, and like Eucalyptus, is a member of Myrtaceae. Furthermore, C. cubensis has been found on Miconia spp. native to South America and residing in Melastomataceae. Recent surveys have yielded C. cubensis isolates from new hosts, characterized in this study based on DNA sequences for the ITS and β-tubulin gene regions. These hosts include native Clidemia sericea and Rhynchanthera mexicana (Melastomataceae) in Mexico, and non-native Lagerstroemia indica (Pride of India, Lythraceae) in Cuba. Isolates from these hosts and areas group in the sub-clade of C. cubensis accommodating the South American collections of the fungus. This sub-clade also includes isolates recently collected from Eucalyptus in Cuba, which are used to epitypify C. cubensis. New host records from Southeast Asia include exotic Tibouchina urvilleana from Singapore and Thailand and native Melastoma malabathricum (Melastomataceae) in Sumatra, Indonesia. Consistent with their areas of occurrence isolates from the latter collections group in the Asian sub-clade of C. cubensis. DNA sequence comparisons of isolates from Tibouchina lepidota in Colombia revealed that they represent a new sub-clade within the greater Chrysoporthe clade. Isolates in this clade are described as Chrysoporthe inopina sp. nov., based on distinctive morphological differences.     

Isolation and characterization of microsatellite loci in Cylindrocladium pauciramosum

Ten polymorphic microsatellite markers were developed for Cylindrocladium pauciramosum, a plant pathogen with a wide host range, which poses a serious problem in South African Eucalyptus nurseries. Polymorphism was evaluated on 43 isolates collected from Colombia and South Africa. Each locus had between three and six alleles. Testing for random mating showed multilocus equilibrium for a population of 40 isolates from a South African forestry nursery. Cross‐species transferability tested for 19 other Cylindrocladium species found amplification only in C. spathulatum, which is phylogenetically closely related to C. pauciramosum.     

Identification of Xanthomonas campestris pv. Juglandis from (Persian) English Walnut Nursery Trees in South Africa

Bacterial isolates producing yellowish colonies on Nutrient Agar were recovered from symptoms of suspect walnut blight disease on leaves of nursery trees in the southwestern Cape Province of South Africa. The isolates were identified by pathogenicity tests on leaves of walnut and plum trees in the greenhouse. Fifteen isolates from four cultivars at two nurseries produced typical lesions of blight on walnut and one isolate. typical lesions of bacterial spot disease on plum leaves. Cluster analysis was done on 28 characteristics recorded from colony growth. colour. form. and elevation on four different culture media, and starch hydrolysis on a semi-selective medium for the isolation of Xanthomonas campestris pv. juglandis. Total DNA of the isolates was digested with restriction endonuclease Spel and resolved by contour-clamped homogeneous electric field (CHEF) electrophoresis. Two phenotypic clusters were distinguished among the 15 South African and one reference strain of X.c.pv. juglandis at the 54%S_sm level. The isolate which induced disease symptoms on plum grouped with reference strains of Xanthomonas campestris pv. pruni in a third cluster. Two-thirds of the isolates were not characterized on the semi-selective medium for X.c. pv. juglandis. DNA restriction fragment banding patterns were similar for most isolates of X.c.juglandis in the same phenotypic cluster. However, DNA banding patterns were non-distinct for some isolates with similar phenotypic characters. Phenotypic characteristics and DNA restriction fragment banding patterns of the isolates were not correlated with geographical origin or cultivar specificity.     

Identification and Molecular Characterizations of Neoscytalidium dimidiatum Causing Stem Canker of Red‐fleshed Dragon Fruit (Hylocereus polyrhizus) in Malaysia

During the year 2008 to 2009, a new disease of stem canker was noticed in most red‐fleshed dragon fruit (Hylocereus polyrhizus) plantations in Malaysia. The symptoms observed were small circular sunken orange spot, black pycnidia and rotted stem. This study was conducted to determine the occurrence of the stem canker on H. polyrhizus in Malaysia, subsequently to isolate, identify and characterize the fungal pathogen based on morphology and molecular characteristics and pathogenicity test. From the surveyed 20 plantations in Malaysia, stem canker was detected in all the plantations. A total of 40 isolates of Scytalidium‐like fungus were isolated and identified as Neoscytalidium dimidiatum based on morphological characteristics and ITS region sequences, which showed 99% similarity to N. dimidiatum (FJ648577). From the phylogenetic analysis using maximum‐likelihood tree, isolates of N. dimidiatum from stem canker of H. polyrhizus were grouped together and did not show any sequence variation. From pathogenicity test, all 40 isolates of N. dimidiatum were pathogenic causing stem canker on H. polyrhizus. To our knowledge, this is the first report of stem canker of H. polyrhizus caused by N. dimidiatum in Malaysia.     

Diversimorbus metrosiderotis gen. et sp. nov. and three new species of Holocryphia (Cryphonectriaceae) associated with cankers on native Metrosideros angustifolia trees in South Africa

The Cryphonectriaceae includes important tree pathogens, especially on the Myrtales. During a routine disease survey in the Western Cape Province of South Africa, a fungus resembling the Eucalyptus pathogen Holocryphia eucalypti was observed on native Metrosideros angustifolia (Myrtales). The aims of this study were to identify the fungus and to expand surveys for fungi in the Cryphonectriaceae on M. angustifolia. Fungi were identified based on DNA sequence comparisons and morphological features, and their pathogenicity was tested on M. angustifolia under field conditions. Based on morphology and multigene phylogenetic analyses of DNA sequence data from six gene regions, we describe a new genus including a single species and three new species of Holocryphia (Cryphonectriaceae) from M. angustifolia. These fungi are provided with the names Diversimorbus metrosiderotis gen. et sp. nov., Holocryphia capensis sp. nov., Holocryphia gleniana sp. nov., and Holocryphia mzansi sp. nov. We also revise H. eucalypti, the type of the genus, to include only isolates from Eucalyptus in South Africa. Research results indicated that H. mzansi may undergo host shifts between different tree genera in the Myrtaceae. Inoculation tests showed that isolates of all the newly described species can cause lesions on the branches of M. angustifolia, indicating that they are all pathogens of this tree.     

DNA bar-coding reveals source and patterns of <Emphasis Type="Italic">Thaumastocoris peregrinus</Emphasis> invasions in South Africa and South America

Thaumastocoris peregrinus is a recently introduced invertebrate pest of non-native Eucalyptus plantations in the Southern Hemisphere. It was first reported from South Africa in 2003 and in Argentina in 2005. Since then, populations have grown explosively and it has attained an almost ubiquitous distribution over several regions in South Africa on 26 Eucalyptus species. Here we address three key questions regarding this invasion, namely whether only one species has been introduced, whether there were single or multiple introductions into South Africa and South America and what the source of the introduction might have been. To answer these questions, bar-coding using mitochondrial DNA (COI) sequence diversity was used to characterise the populations of this insect from Australia, Argentina, Brazil, South Africa and Uruguay. Analyses revealed three cryptic species in Australia, of which only T. peregrinus is represented in South Africa and South America. Thaumastocoris peregrinus populations contained eight haplotypes, with a pairwise nucleotide distance of 0.2–0.9% from seventeen locations in Australia. Three of these haplotypes are shared with populations in South America and South Africa, but the latter regions do not share haplotypes. These data, together with the current distribution of the haplotypes and the known direction of original spread in these regions, suggest that at least three distinct introductions of the insect occurred in South Africa and South America before 2005. The two most common haplotypes in Sydney, one of which was also found in Brisbane, are shared with the non-native regions. Sydney populations of T. peregrinus, which have regularly reached outbreak levels in recent years, might thus have served as source of these three distinct introductions into other regions of the Southern Hemisphere.     

Characterization of Colletotrichum kahawae Isolates Causing Coffee Berry Disease in Angola

Coffee Berry Disease, caused by Colletotrichum kahawae, is a major limitation for Arabica coffee cultivation in Africa and for which genetic control is only partially effective. As part of the effort to re‐launch coffee cultivation in Angola, our aim was to study the diversity of this pathogen and so contribute to more effective breeding for disease resistance. A collection of 30 C. kahawae isolates showed limited diversity in genetic and colony characters. However, some isolates are distinct, suggesting that breeding for disease resistance in Angola should be dependent on an adequate knowledge of the diversity of local and neighbouring C. kahawae isolates. Analysis of C. kahawae nrDNA nucleotide sequences showed distinct lineages clustering within the broad diversity of C. gloeosporioides, prompting further studies aimed at understanding the origin and pathogenic specialization of C. kahawae.     

Sporothrix eucalypti (sp. nov.), a shoot and leaf pathogen ofEucalyptus in South Africa

A species ofSporothrix was consistently isolated from leaf spots and serious shoot infections on a clone ofEucalyptus grandis in Northern Natal, South Africa. The fungus was morphologically distinct from other species in the genus and is consequently described as a new taxon,S. eucalypti. Sporothrix eucalypti was shown to be highly virulent in pathogenicity tests on a number ofE. grandis clones. Significant differences amongst susceptibility of clones were also detected in these tests.Sporothrix eucalypti represents a new pathogen ofEucalyptus that has the potential to cause substantial damage to this host in South Africa and probably elsewhere in the world.     

Characterization of emerging populations of Fusarium oxysporum f. sp. conglutinans causing cabbage wilt in China

Fusarium oxysporum f. sp. conglutinans (FOC) causes Fusarium wilt, a disease of cabbage that has brought about significant economic loss throughout northern China since it was first detected in 2001. To characterize the Chinese FOC isolates, we compared the cultural characteristics, pathogenicity and races between the Chinese isolates and the type strains (race 1: 52,557 and race 2: 58,385). The Chinese FGL‐03‐6 isolate had cultural characteristics similar to those of strain 52,557, including colony growth rate, colony and spore characteristics and responses to temperature changes, while the strain 58,385 grew faster, produced more pigment and spores and was more adaptable to temperature fluctuations. The lethal temperature for all strains was 60°C, and the optimal temperatures for pathogen growth on potato dextrose agar and pathogenicity on plants were 25°C and 25 to 30°C, respectively. Tests for race and pathogenicity indicated that different cabbage cultivars had similar resistance reactions to FGL‐03‐6 and 52,557. However, the pathogenicity of FGL‐03‐6 was similar to that of 58,385, which infected quickly and caused more severe disease symptoms. This study further provides information regarding characterizing different strains of F. oxysporum f. sp. conglutinans.     

Novel Cryphonectriaceae from La Réunion and South Africa,and their pathogenicity on <Emphasis Type="Italic">Eucalyptus</Emphasis>

Fungi in the Cryphonectriaceae are important canker pathogens of plants in the Melastomataceae and Myrtaceae (Myrtales). These fungi are known to undergo host jumps or shifts. In this study, fruiting structures resembling those of Cryphonectriaceae were collected and isolated from dying branches of Syzygium cordatum and root collars of Heteropyxis natalensis in South Africa, and from cankers on the bark of Tibouchina grandifolia in La Réunion. A phylogenetic species concept was used to identify the fungi using partial sequences of the large subunit and internal transcribed spacer regions of the nuclear ribosomal DNA, and two regions of the β-tubulin gene. The results revealed a new genus and species in the Cryphonectriaceae from South Africa that is provided with the name Myrtonectria myrtacearum gen. et sp. nov. Two new species of Celoporthe (Cel.) were recognised from La Réunion and these are described as Cel. borbonica sp. nov. and Cel. tibouchinae sp. nov. The new taxa were mildly pathogenic in pathogenicity tests on a clone of Eucalyptus grandis. Similar to other related taxa in the Cryphonectriaceae, they appear to be endophytes and latent pathogens that could threaten Eucalyptus forestry in the future.     

Molecular characterization and pathogenicity of Colletotrichum sp. from guava

Guava (Psidium guajava) fruit is vulnerable to postharvest diseases, such as anthracnose. In the present study, molecular characterisation and pathogenicity of Colletotrichum associated with antharcnose disease of guava fruit were conducted. From anthracnose lesion of guava, 20 isolates were successfully recovered. Based on colony colours, conidia, appressoria and presence or absence of setae, and ITS regions and ß-tubulin gene sequences, the isolates were identified as Colletotrichum gloeosporioides. Phylogenetic analysis based on combined data-sets using neighbour-joining method showed that C. gloeosporioides isolates did not group with C. gloeosporioides epitype strain, and thus the isolates were referred to as C. gloeosporioides species complex or C. gloeosporioides sensu lato. Pathogenicity tests using wounded treatment showed that C. gloeosporioides isolates from guava were pathogenic causing anthracnose on the fruits. The present study showed that C. gloeosporioides sensu lato is the most common species causing antharcnose disease of guava fruit.     

Presence of Leptosphaeria maculans Group A and Group B Isolates in Sweden

Leptosphaeria maculans isolates have been assigned to one of two groups, A or B, on the basis of differences in their characteristics. Group A can further be divided into pathogenicity groups (PG) 2, 3 and 4 and group B into PG1. To determine if isolates belonging to the aggressive canker forming group A are present in Sweden, physiological and genetic characterisation of 120 isolates collected in the year 2000 were performed. Thirty‐seven isolates were classified as belonging to pathogenicity group PG3 and 63 isolates as PG4, based on a cotyledon assay. Twenty isolates did not cause any symptoms at all, and were classified as PG1. When comparing two geographical regions, Skåne and Östergötland, equal numbers of PG3 and PG4 isolates were found. By analysing the isolates by PCR, the collection was further classified into 100 group A isolates and 20 group B isolates. A corresponding classification of the isolates was observed when the ability to produce pigments in Czapek Dox broth was examined. The results showed a clear predominance of group A. This was also the case for the isolate collection from 2001. In a detailed survey of disease development in a L. maculans infected winter oilseed rape field in southern Sweden (Skåne), basal stem canker was not observed until early June Although the disease index value increased from 8.4 in June to 18.0 in July, few severely damaged plants were observed before harvest in mid‐July, despite infection with group A isolates.     

Development and characterization of microsatellite loci for the tropical tree pathogen Botryosphaeria rhodina

Nineteen simple sequence repeat (SSR) markers were developed for the pathogen and blue stain fungus Botryosphaeria rhodina. Eight pairs were found to be polymorphic among isolates collected from Pinus spp., whereas a further five pairs were polymorphic when isolates from Pinus spp. were compared with those from Eucalyptus spp. Nine isolates of B. rhodina collected from pines and eucalypts in South Africa, Mexico and Indonesia were used to demonstrate the range of the markers. Testing the markers yielded preliminary evidence that relationships among isolates are more closely linked to host than to geographical origin.     

I. Pathogenic variation in fungi and bacteria and mycorrhizal compatibility: Variation in Pseudomonas morsprunorum and the selection of cherry cultivars resistant to bacterial canker

During 1971 and 1972 leaf spot infection caused by Pseudomonas morsprunorum was far more severe on the cv. Roundel than on the normally more susceptible cv. Napoleon, and Roundel supported higher leaf surface populations of the pathogen. This unprecedented reversal in the established field performance of the two cultivars was associated with the presence on the trees of a colony variant (race 2) of P. morsprunorum that differed from the forms of the organism previously described (race 1) in physiological, pathological and phage sensitivity characteristics. In inoculation experiments race 2 isolates showed some specificity for Roundel and race 1 isolates for Napoleon. This difference was reflected in the distribution of the races on trees of the two cultivars growing in experimental plots and commercial orchards. Similar interactions between race and host genotype were observed amongst progeny from breeding programmes and material originally selected for resistance to bacterial canker after inoculation with race 1 proved susceptible to race 2. Three distinct groups of isolates with the colony characteristics of race 2 have been identified and one of these has affinities with Pseudomonas syringae. Group 1 isolates, corresponding to P. morsprunorum and comprising non-fluorescent forms, were the most virulent in pathogenicity tests and accounted for the majority of race 2 infections in the field.     

Morphological and Genetic Analyses of Hellebore Leaf Spot Disease Isolates from Different Geographic Origins Show Low Variability and Reveal Molecular Evidence for Reclassification into Didymellaceae

Hellebore leaf spot, caused by Coniothyrium hellebori, is the most common fungal disease of Helleborus species not only in botanical and ornamental gardens but also in nurseries. To correct the current lack of knowledge regarding this widely distributed pathogen, this study investigated 25 C. hellebori isolates collected from different countries in North America and Europe, primarily Germany. The morphology, pathogenicity and molecular genetic relationships on the basis of random amplified polymorphic DNA (RAPD) of these isolates were studied. RAPD primers produced a total of 394 bands, of which 40% were polymorphic. Genetic distances were calculated, and a dendrogram with bootstrap analysis was constructed by the unweighted pair group method with arithmetic mean (UPGMA) cluster method. All isolates were identified as C. hellebori, the causal agent of the disease. Two C. hellebori subclades were found, which could not be correlated with the geographic origin of the isolate, but with the plant host species and morphological characteristics. Sequence comparisons of the large subunit and internal transcribed spacer loci between C. hellebori and sequences from GenBank revealed that C. hellebori has to be grouped into the Didymellaceae family and rather belongs to Phoma or Microsphaeropsis than to Coniothyrium. This work represents the first study of this plant pathogen causing severe damage in Helleborus stocks and provides important information for the development of future Helleborus resistance breeding strategies.     

Floral colour variation of Nicotiana glauca in native and non-native ranges: Testing the role of pollinators' perception and abiotic factors

• Invasive plants displaying disparate pollination environments and abiotic conditions in native and non-native ranges provide ideal systems to test the role of different ecological factors driving flower colour variation.
• We quantified corolla reflectance of the ornithophilous South American Nicotiana glauca in native populations, where plants are pollinated by hummingbirds, and in populations from two invaded regions: South Africa, where plants are pollinated by sunbirds, and the Balearic island of Mallorca, where plants reproduce by selfing. Using visual modelling we examined how corolla reflectance could be perceived by floral visitors present in each region. Through Mantel tests we assessed a possible association between flower colour and different abiotic factors.
• Corolla reflectance variation (mainly along medium to long wavelengths, i.e. human green-yellow to red colours) was greater among studied regions than within them. Flower colour was more similar between South America and South Africa, which share birds as pollinators. Within invaded regions, corolla reflectance variation was lower in South Africa, where populations could not be distinguished from each other by sunbirds, than in Spain, where populations could be distinguished from each other by their occasional visitors. Differences in corolla colour among populations were partially associated with differences in temperature.
• Our findings suggest that shifts in flower colour of N. glauca across native and invaded ranges could be shaped by changes in both pollination environment and climatic factors. This is the first study on plant invasions considering visual perception of different pollinators and abiotic drivers of flower colour variation.