Teresa A. Coutinho

Eucalyptus rust: A disease with the potential for serious international implications

Eucalyptus spp. are propagated extensively as non-natives in plantations in many parts of the tropics and sub-tropics. A number of diseases result in serious losses to this economically important forest resource. Eucalyptus rust, caused by Puccinia psidii, is one such example. The economic losses due to this disease are the result of infections of seedlings, young trees, and coppice. P. psidii occurs predominately in Central and South America, but reports of a similar rust are known from other areas. Eucalyptus rust is a remarkable disease in that the pathogen is not known on eucalypts in their centers of origin. It has apparently originated on native Myrtaceae in South America and is highly infective on some Eucalyptus spp. planted there. P. psidii causes one of the most serious forestry diseases in Brazil and is considered to be the most serious threat to eucalypt plantations worldwide. Advances in eucalyptus rust research are reviewed here, with a focus on topics such as distribution, host range, pathogen specialization, symptomatology, etiology, epidemiology, and control.

Sphaeropsis sapinea and Botryosphaeria dothidea endophytic in Pinus spp. and Eucalyptus spp. in South Africa.

Sphaeropsis sapinea (Fr.: Fr.) Dyko & B. Sutton and the anamorph of Botryosphaeria dothidea (Moug.) Ces. et De Not. are morphologically and ecologically similar fungi that cause serious canker and die-back diseases of Pinus and Eucalyptus spp. respectively in South Africa. In this article, the presence of both these fungi as symptomless endophytes In healthy pine and eucafypt tissue was demonstrated. Sphaeropsis sapinea was present in 50% of young, green P patula Schl. et Cham., and 90% of P. radiata D. Don cones. In contrast, it was virtually absent from the cones of P. elliottii Engalm. et Vasey and P. taeda L. Botryosphaeria dothidea, on the other hand, was found to be common in all the Eucalyptus spp. tested, occurring in 93% of E. smithii R. T. Bak., 77% of E. camaldulensis Dehnh., 63% of E. grandis Hill ex Maid, and 57% of E. nitens (Deane et Maid.) Maid, leaves tested. The enigma of the rapid Ingress of both these fungi In stressed or damaged trees might therefore be explained by their endophytic habit.

Pitch canker caused by Fusarium circinatum – a growing threat to pine plantations and forests worldwide

Pitch canker, caused by the fungus Fusarium circinatum, is one of the most important pathogens of Pinus species. Sporadic outbreaks and epidemics caused by this fungus have been reported from numerous countries. Symptoms differ depending on the host species, geographical region, climatic conditions and associated insects. Pitch canker represents a significant threat to countries where non-native and susceptible Pinus spp. are grown intensively in plantations.Athorough understanding of the ecology and epidemiology of the causal agent is an important prerequisite to managing this threat. The aim of this review is to summarise contemporary knowledge relating to the pitch canker pathogen, with a particular focus on its threat to plantation forestry.

PCR-based identification of MAT-1 and MAT-2 in the Gibberella fujikuroi species complex.

ABSTRACT All sexually fertile strains in the Gibberella fujikuroi species complex are heterothallic, with individual mating types conferred by the broadly conserved ascomycete idiomorphsMAT-1 and MAT-2. We sequenced both alleles from all eight mating populations, developed a multiplex PCR technique to distinguish these idiomorphs, and tested it with representative strains from all eight biological species and 22 additional species or phylogenetic lineages from this species complex. In most cases, either an ∼800-bp fragment from MAT-2 or an ∼200-bp fragment from MAT-1 is amplified. The amplified fragments cosegregate with mating type, as defined by sexual cross-fertility, in a cross of Fusarium moniliforme (Fusarium verticillioides). Neither of the primer pairs amplify fragments from Fusarium species such as Fusarium graminearum, Fusarium pseudograminearum, andFusarium culmorum, which have, or are expected to have,Gibberella sexual stages but are thought to be relatively distant from the species in the G. fujikuroi species complex. Our results suggest that MAT allele sequences are useful indicators of phylogenetic relatedness in these and otherFusarium species.

Pantoea ananatis: an unconventional plant pathogen

UNLABELLED Pantoea ananatis causes disease symptoms in a wide range of economically important agricultural crops and forest tree species worldwide. It is regarded as an emerging pathogen based on the increasing number of reports of diseases occurring on previously unrecorded hosts in different parts of the world. Its unconventional nature lies in the fact that, unlike the majority of plant pathogenic microbes, P. ananatis is capable of infecting humans and occurs in diverse ecological niches, such as part of a bacterial community contaminating aviation jet fuel tanks and contributing to growth promotion in potato and pepper. TAXONOMY Bacteria; Gammaproteobacteria; family Enterobacteriaceae; genus Pantoea. MICROBIOLOGICAL PROPERTIES Gram-negative; facultatively anaerobic; most strains are motile and produce a yellow pigment in culture; indole positive. BIOLOGY: Pantoea ananatis is a common epiphyte; it also occurs endophytically in hosts where it has been reported to cause disease symptoms and in hosts where no such symptoms have been described. Some strains are ice-nucleating, a feature which has been used as a biological control mechanism against some insect pests of agricultural crops and by the food industry. DISEASE SYMPTOMS Pantoea ananatis infects both monocotyledonous and dicotyledonous plants. The symptoms are diverse depending on the host infected, and include leaf blotches and spots, die-back, and stalk, fruit and bulb rot. BIOLOGICAL CONTROL AGENT: Pantoea ananatis has both antifungal and antibacterial properties. These characteristics have the potential of being exploited by biological control specialists.

The root rot fungus Armillaria mellea introduced into South Africa by early Dutch settlers.

Dead and dying oak (Quercus) and numerous other woody ornamental trees and shrubs showing signs and symptoms of Armillaria root rot were identified in the Company Gardens, Cape Town, South Africa, which were established in the mid‐1600s by the Dutch East Indies Trading Company. Nineteen isolates from dying trees or from mushrooms were collected and analysed to identify and characterize the Armillaria sp. responsible for the disease. The AluI digestion of the amplified product of the first intergenic spacer region (IGS‐1) of the rRNA operon of 19 isolates from the Company Gardens was identical to that of some of the European isolates of A. mellea s. s. The IGS‐1 region and the internal transcribed spacers (ITS) were sequenced for some of the Cape Town isolates. Phylogenetic analyses placed the Cape Town isolates in the European clade of A. mellea, which is distinct from the Asian and North American clades of this species. Identification based on sexual compatibility was conducted using A. mellea tester strains in diploid–haploid pairings, which showed some compatibility between the Cape Town isolates and testers from Europe. Somatic compatibility tests (diploid–diploid pairings) and DNA fingerprinting with multilocus, microsatellite probes indicated that the Cape Town isolates were genetically identical and may have resulted from vegetative (clonal) spread from a single focus in the centre of the original Company Gardens (c. 1652). The colonized area is at least 345 m in diameter. Assuming a linear spread rate underground of 0.3 m/year to 1.6 m/year, the genet (clone) was estimated to be between 108 and 575 years old. These data suggest that A. mellea was introduced into Cape Town from Europe, perhaps on potted plants, such as grapes or citrus, planted in the Company Gardens more than 300 years ago.

Botryosphaeria eucalyptorum sp. nov., a new species in the B. dothidea -complex on Eucalyptus in South Africa.

Two morphologically similar fungi are as- sociated with canker and die-back of eucalypts in South Africa, one of which was identified as part of the Botryosphaeria dothidea-complex. In this study, the identity of the other fungus was determined by com- paring morphology, pathogenicity and DNA se- quence analysis of isolates of both taxa. Based on these results, a new species, Botryosphaeria eucalypto- rum, and its anamorph Fusicoccum eucalyptorum, are described. Although the teleomorph is morphologi- cally similar to other taxa in the B. dothidea-complex, conidial characteristics of the anamorph are distinct, as well as the sequences of the nrDNA internal tran- scribed spacers ITS1 and ITS2. Like B. dothidea, the fungus is pathogenic to Eucalyptus, although there do not appear to be clear differences in pathogenic- ity between these two species.

Multiple gene genealogies and phenotypic data reveal cryptic species of the Botryosphaeriaceae: A case study on the Neofusicoccum parvum/N. ribis complex

Neofusicoccum parvum and N. ribis (Botryosphaeriaceae, Ascomycetes) are closely related, plant pathogenic fungi with a world-wide distribution on a wide range of woody hosts. Species boundaries in the N. parvum/N. ribis complex have eluded definition, despite the application of various tools for characterisation. In this study, we test the hypothesis that only one species exists amongst isolates from the N. parvum/N. ribis complex, identified from Syzygiumcordatum trees across their native distribution in South Africa. Genealogical concordance phylogenetic species recognition (GCPSR) was applied based on concordance of genealogies obtained from DNA sequence data for five nuclear loci. These data showed that the single species hypothesis must be rejected. Rather, all analyses support the existence of three previously unrecognised, cryptic species within the N. parvum/N.ribis complex from S. cordatum, in addition to N. parvum and N. ribis. The three lineages reflecting these cryptic taxa are sympatric across their geographical range, indicating barriers to gene flow other than geographic isolation. Phenotypic characters failed to detect all the species uncovered by the GCPSR. Sequence data of the Internal Transcribed Spacer (ITS) of the ribosomal DNA locus, which is thought to be useful for barcoding in fungi, did not distinguish all the species with confidence. RNA polymerase II subunit (RPB2) was the most informative to distinguish all the species a posteriori to the application of GCPSR. The results reflect the critical importance of using multiple gene genealogies and adequate sampling to identify cryptic species and to characterise the true diversity within the Botryosphaeriaceae.

Bacterial Blight and Dieback of Eucalyptus Species, Hybrids, and Clones in South Africa

During 1998, a new disease appeared on trees representing a Eucalyptus grandis × E. nitens (GN) hybrid in a nursery in KwaZulu/Natal. The disease has subsequently spread to other Eucalyptus species, hybrids, and clones in nurseries and plantations throughout South Africa. Typical symptoms of the disease include dieback of young shoots and leaf blight. This ultimately leads to stunting of trees. The objective of this study was to isolate and identify the causal agent of the disease. A bacterium was consistently isolated from infected tissue. Pathogenicity tests were undertaken with a range of bacterial strains. Four pathogenic strains were selected from different geographical regions and Eucalyptus hosts for further study. The bacterium causing Eucalyptus leaf and shoot blight is gram negative and rod-shaped, varying in size from 0.5 to 0.75 μm wide and 1.0 to 2.0 μm long. Colonies of this bacterium have a yellow pigment. The results from the Biolog tests identified the bacterium as Pantoea agglomerans with a similarity index of 0.315. The 16S rDNA sequences of the purported Pantoea sp. were compared with those of other related Enterobacteriaceae from GenBank/EMBL. Phylogenetic analysis using PAUP revealed that the isolates group together with P. agglomerans, P. ananatis, and P. stewartii subsp. stewartii. The fatty acid profiles and phenotypic characteristics of the new pathogen are similar to P. ananatis, and % G + C is within the range of this species. DNA:DNA hybridization between the four strains and the type strain of P. ananatis conclusively showed that the bacterium causing blight and dieback of Eucalyptus in South Africa belongs to this species. This is the first report in which P. ananatis has been found as a causal agent of a disease on Eucalyptus.

Pantoea vagans sp. nov., Pantoea eucalypti sp. nov., Pantoea deleyi sp. nov. and Pantoea anthophila sp. nov.

Bacteria isolated from eucalyptus leaves and shoots showing symptoms of blight and die-back collected in Uganda, Uruguay and Argentina and from maize displaying brown stalk rot symptoms in South Africa were tentatively placed in the genus Pantoea on the basis of phenotypic and biochemical tests. These isolates, together with two strains (LMG 2558 and LMG 2560) previously assigned to Pantoea agglomerans based on protein electrophoregrams but later excluded from this species, were further investigated using molecular techniques. 16S rRNA gene sequencing and multilocus sequence analyses (MLSA) revealed that the strains were phylogenetically closely related to Pantoea agglomerans, Pantoea stewartii and Pantoea ananatis. MLSA and amplified fragment length polymorphism analysis placed the strains into four separate clusters, not containing any of the type strains of species of the genus Pantoea. DNA-DNA hybridization confirmed the classification of the isolates into four novel species, for which the names Pantoea vagans sp. nov. (type strain R-21566T=LMG 24199T=BCC 105T=BD 765T), Pantoea eucalypti sp. nov. (type strain R-25678T=LMG 24197T=BCC 076T=BD 769T), Pantoea deleyi sp. nov. (type strain R-31523T=LMG 24200T=BCC 109T=BD 767T) and Pantoea anthophila sp. nov. (type strain LMG 2558T=BD 871T=NCPPB 1682T) are proposed.