Nursery‐linked plantation outbreaks and evidence for multiple introductions of the pitch canker pathogen Fusarium circinatum into South Africa

In recent years, Pinus plantation forestry has been significantly hampered by outbreaks of pitch canker caused by the fungus Fusarium circinatum. This study investigated the role of Pinus host, geographic origin and reproductive mode in structuring the F. circinatum populations in plantations. For this purpose, 159 isolates originating from diseased plantation trees in the Western and Eastern Cape Provinces of South Africa were genotyped using 10 microsatellite markers. Analyses of these data revealed 30 multilocus haplotypes and that the populations were distinct based on geographic origin as well as host. However, shared haplotypes were observed between populations, showing that these populations are connected, possibly through the movement of haplotypes. A second aim was to determine whether the genetic variation found in these populations of the fungus could be attributed to outbreaks of the seedling disease caused by this pathogen in Pinus nurseries. To achieve this goal, an additional set of 43 isolates originating from pine seedling nurseries was genotyped and analysed. The results showed that the populations of F. circinatum in plantations most probably originated from the nursery outbreaks that occurred prior to the plantation outbreak. Inferences regarding reproductive mode further showed that sexual reproduction has little impact on the genetic makeup of the F. circinatum populations and that they primarily reproduce asexually. Overall, the results of this study showed that the F. circinatum diversity in South Africa has arisen due to multiple introductions of the pathogen and is not due to sexual reproduction.     

Genotypic diversity in a South African population of the pitch canker fungus Fusarium subglutinans f.sp. pini

The genotypic diversity in a South African population of Fusarium subglutinans f.sp. pini ( F.s. pini ) was determined, based on the number of vegetative compatibility groups (VCGs). Isolates of F.s. pini from South Africa (69), California (five) and Florida (19) were included in the study. The nit1 (or nit3 ) and NitM mutants were selected as chlorate resistant sectors and paired on minimal medium. The South African isolates of F.s. pini were assigned to 23 different VCGs. No heterokaryons formed between isolates from South Africa, California and Florida. The high degree of genotypic diversity in the South African population of F.s. pini is probably due to some level of sexual reproduction in the population.     

Evidence for a new introduction of the pitch canker fungus Fusarium circinatum in South Africa

Fusarium circinatum causes pitch canker of Pinus species in many parts of the world. The fungus was first recorded in South Africa in 1990 as a pathogen of P. patula seedlings and emerged later as a pathogen of established plantation trees, especially P. radiata in the Western Cape Province (WCP). In this study the population biology of F. circinatum in the WCP was explored. The aim was to determine the possible origin and reproductive mode of the pathogen, with the ultimate intention of informing disease management strategies in the region. Vegetative compatibility assays, sexual mating studies and amplified fragment length polymorphism analyses were used. For comparative purposes, an isolate collection obtained from diseased P. radiata seedlings in a commercial nursery in the region, as well as a set of isolates from commercial seedling nurseries in the central and northern parts of South Africa, were included. The results showed that the WCP population of F. circinatum employs a predominantly asexual mode of reproduction and that it is highly differentiated from populations of the fungus elsewhere in South Africa. However, limited genetic structure was found within the respective WCP isolate collections. Overall these findings suggest that pitch canker in the WCP originates from one or more separate introductions of the pathogen and that its movement in the region is not restricted. More effective strategies are thus required to limit and manage the effects of F. circinatum in plantations in this region of South Africa.     

Unique clones of the pitch canker fungus, <Emphasis Type="Italic">Fusarium circinatum,</Emphasis> associated with a new disease outbreak in South Africa

Pitch canker of pines is caused by the fungus Fusarium circinatum. In South Africa, this pathogen has mostly been a nursery problem. From 2005, however, outbreaks of pitch canker have been reported from established Pinus radiata and P. greggii in the Western and Eastern Cape Provinces. Most recently, pitch canker-like symptoms were observed on 10-year-old P. greggii trees in a plantation in the midlands of the KwaZulu-Natal (KZN) Province. The aim of this study was to: (i) identify the causal agent of the observed symptoms, (ii) determine the genetic diversity, and (iii) the mode of reproduction of this fungal population. Furthermore, the aggressiveness of isolates from these trees was compared with that of isolates obtained previously from P. patula in South Africa. Isolates from the P. greggii trees in KZN were confirmed as F. circinatum based on both morphology and DNA sequence analyses. Microsatellite marker analyses revealed the presence of five genotypes of F. circinatum, not previously reported from other plantations in South Africa, with one of these genotypes being dominant. These genotypes were all pathogenic to P. patula and P. elliottii. No evidence of sexual reproduction was detected in the KZN population of the fungus. This was consistent with the fact that isolates from P. greggii were all of the MAT-2 mating type, in contrast to previously collected isolates from across South Africa that included both mating types. The results suggest that the outbreak of pitch canker on P. greggii in KZN represents a separate introduction of F. circinatum into the region with important implications for managing the disease.     

Factors influencing infection of mechanical wounds by Fusarium circinatum on Monterey pines (Pinus radiata)

Pitch canker, caused by the fungus Fusarium circinatum , is a disease affecting many pine tree species. In California, Pinus radiata (Monterey pine) is the principal pine host affected by pitch canker. This investigation into factors affecting infection frequency by F. circinatum of P. radiata examined the influence of: (i) wound size; (ii) relative humidity; (iii) time of inoculation; (iv) inoculum density; and (v) wound age. Wounded branches sustained significantly more infections when large-diameter (1·6 mm) rather than small-diameter (0·5 mm) wounds were made. Infection frequencies tended to be higher at 100% RH than at ambient humidity, although these differences were not statistically significant. Infection frequencies were significantly higher on branches inoculated after 17·00 h than on branches inoculated before noon. Infection frequencies were significantly higher for wounded branches spray-inoculated with 5 × 10⁷ rather than 1 × 10⁷ spores mL⁻¹. Infection frequencies of pruning wounds declined as wounds aged.     

High diversity and clonality are hallmarks of Fusarium circinatum in South Africa

Pitch canker on plantation-grown Pinus species, caused by the fungal pathogen Fusarium circinatum, first appeared in the western and southern Cape regions of South Africa. However, outbreaks have subsequently been reported from the major plantation growing regions of KwaZulu-Natal and Limpopo in the eastern, summer rainfall regions of the country. It is more than 10 years since the last detailed population genetics studies on F. circinatum in the region were conducted. To shed light on the population biology of F. circinatum in this region of South Africa, we used microsatellite markers and mating-type assays to study a collection of 296 isolates from different nurseries and plantation sites. Our results showed that populations in the region are highly diverse, but strongly interconnected, with various genotypes shared across nursery and plantation collection sites. In contrast to nursery populations, those associated with pitch canker outbreaks were characterized by the presence of relatively small numbers of dominant genotypes that were generally widespread across the region. Opposite mating-type individuals occurred in most of the isolate collections, but multilocus linkage disequilibrium analyses pointed towards clonality being the main reproductive mode of F. circinatum in the region. Most of the pathogen's genetic variation could probably have resulted from multiple different introductions into the country and more specifically, into the summer rainfall region. Because the spread and establishment of invasive pathogens are typically driven by aggressive clones, the results of this study provide important considerations for current and future Pinus disease management strategies.     

Pitch canker disease of pines

ABSTRACT Pitch canker, caused by Fusarium circinatum, is a disease affecting pines in many locations throughout the world. The pathosystem was originally described in the southeastern (SE) United States and was identified in California in 1986. Limited vegetative compatibility group (VCG) diversity in the California population of F. circinatum, relative to the SE United States, suggests the former is a recently established and clonally propagating population. Although the much greater VCG diversity found in the SE United States is suggestive of out-crossing, molecular markers indicate that many vegetatively incompatible isolates are clonally related. This implies that VCG diversity may derive, at least in part, from somatic mutations rather than sexual reproduction. Pitch canker is damaging to many pine species and one at particular risk is Monterey pine (Pinus radiata), which is widely grown in plantations and is highly susceptible to pitch canker. However, some Monterey pines are resistant to pitch canker and some severely diseased trees have been observed to recover. The absence of new infections on these trees reflects the operation of systemic induced resistance, apparently in response to repeated infection by the pitch canker pathogen.     

Alternative species to replace Monterey pine plantations affected by pitch canker caused by Fusarium circinatum in northern Spain

The pitch canker pathogen Fusarium circinatum was first found to cause damage in nurseries and pine plantations in northern Spain in 2004. Since then, establishment of pine plantations in the region has decreased as a result of the prohibitions placed on planting Pinus spp. and Pseudotsuga menziesii in areas affected by the disease. However, although most pine species have been found to be susceptible to the pathogen under nursery conditions, little is known about how the fungus affects the trees in the field. Furthermore, it is not known whether some of the native or exotic species commonly planted in the area are also susceptible to F. circinatum. The aim of this study was to evaluate the susceptibility of several conifer species commonly planted in northern Spain to the pitch canker pathogen. For this purpose, two different trials were carried out, one under controlled laboratory conditions and the other in the field. Although most of the conifers were affected by the pathogen in the laboratory tests, only Pinus radiata, Pinus nigra, Pinus pinaster and Pinus uncinata were susceptible to the pathogen in the field.     

Population differentiation in the banana leaf spot pathogen Mycosphaerella musicola, examined at a global scale

Single-copy restriction fragment length polymorphism (RFLP) markers were used to determine the genetic structure of the global population of Mycosphaerella musicola , the cause of Sigatoka (yellow Sigatoka) disease of banana. The isolates of M. musicola examined were grouped into four geographic populations representing Africa, Latin America and the Caribbean, Australia and Indonesia. Moderate levels of genetic diversity were observed for most of the populations ( H  = 0·22–0·44). The greatest genetic diversity was found in the Indonesian population ( H =  0·44). Genotypic diversity was close to 50% in all populations. Population differentiation tests showed that the geographic populations of Africa, Latin America and the Caribbean, Australia and Indonesia were genetically different populations. Using F _ST tests, very high levels of genetic differentiation were detected between all the population pairs ( F _ST > 0·40), with the exception of the Africa and Latin America-Caribbean population pair. These two populations differed by only 3% ( F _ST = 0·03), and were significantly different ( P  < 0·05) from all other population pairs. The high level of genetic diversity detected in Indonesia in comparison to the other populations provides some support for the theory that M. musicola originated in South-east Asia and that M. musicola populations in other regions were founded by isolates from the South-east Asian region. The results also suggest the migration of M. musicola between Africa and the Latin America-Caribbean region.     

Susceptibility of Douglas fir (Pseudotsuga menziesii) to pitch canker, caused by Gibberella circinata (anamorph = Fusarium circinatum)

For better characterization of the risk of pitch canker (caused by Gibberella circinata , anamorph =  Fusarium circinatum ) to Douglas fir ( Pseudotsuga menziesii ), Californian isolates, selected exotic isolates, and ascospore progeny of a cross between wild-type Californian isolates were tested for aggressiveness to this host species. In addition, seedlings from representative provenances of P. menziesii in California were tested for susceptibility to pitch canker. The results revealed only minor differences between isolates, but differences in susceptibility between trees were often significant. The majority of the tested trees were relatively resistant as indicated by the development of only very short lesions, but some were clearly susceptible.     

Population structure and possible origin of Amylostereum areolatum in South Africa

The woodwasp, Sirex noctilio , and its symbiotic fungus, Amylostereum areolatum , cause extensive damage to pine plantations in the Southern Hemisphere. S. noctilio was first reported from South Africa in 1994. In this study, the population diversity of A. areolatum isolates from South Africa, South America, Australasia and Europe was determined by vegetative incompatibility testing. All 108 South African and 26 South American isolates belonged to the same vegetative compatibility group (VCG). This VCG showed a weak incompatibility reaction with the single Tasmanian and single New Zealand isolates tested. This VCG differed from VCGs from Europe. It also differed from isolates associated with the biocontrol nematode, Deladenus siricidicola , which is produced in Australia. It is concluded that the South African and South American populations of A. areolatum share a common origin .     

Characterization of the genetic variation and fungicide resistance in Botrytis cinerea populations on rooibos seedlings in the Western Cape of South Africa

Botrytis cinerea causes severe losses of rooibos seedlings in the Clanwilliam region of the Western Cape of South Africa. A total of 207 isolates were collected from diseased seedlings representing five nurseries. The cryptic species status of these isolates was determined with restriction enzyme analysis of the Bc-hch gene with 206 of the isolates identified as B. cinerea and one isolate as B. pseudocinerea. Analysis of the B. cinerea populations using seven microsatellite loci revealed a high total gene diversity (H), with a mean of 0.67. Analysis of molecular variance indicated that 97 % of the total genetic variation is distributed within subpopulations. Genotype flow was evident between nurseries indicated by shared occurrence of clonal lineages, in agreement with the relatively low but significant population differentiation between nurseries (mean F _ST?=?0.030, P?=?0.001). The distribution of the mating type alleles MAT1-1 and MAT1-2 differed significantly from the ratio of 1:1 for the total population and in two of the five nursery subpopulations. Together with significant linkage disequilibrium found, populations appear to mainly reproduce asexually. Fungicide resistance frequency against iprodione for 198 of the genotyped isolates, showed highly varying levels of resistance between the nurseries. The mean incidence of resistance towards iprodione was 44 % in total, ranging from 0 % to 81 % for the five nurseries. This study has shown an adaptive capacity to overcome current chemical means of control in the studied B. cinerea population.     

Genetic diversity in the mango malformation pathogen and development of a PCR assay

Malformation is a destructive disease of mango, Mangifera indica . Its causal agent possesses the morphological features of Fusarium subglutinans , a species whose taxonomy and nomenclature has recently been in a state of flux. Genetic diversity was examined among 74 F. subglutinans -like isolates from malformed mango in Brazil, Egypt, Florida (USA), India, Israel and South Africa. With nitrate-nonutilizing ( nit ) auxotrophic mutants, seven vegetative compatibility groups (VCGs) were identified. Three of the VCGs were found in a single country, and VCG diversity was greatest in Egypt and the USA where, respectively, four and three different VCGs were found. RAPD profiles generated with arbitrary decamer primers were variable among isolates in different VCGs, but were generally uniform for isolates within a VCG. In PCR assays, a 20-mer primer pair that was developed previously to identify F. subglutinans from maize (mating population [MP]-E of the Gibberella fujikuroi complex) also amplified a specific 448 bp fragment for isolates of F. sacchari from sugarcane (MP-B) and what was probably F. circinatum (pine, MP-H). With the exception of three isolates from Brazil, it did not amplify the fragment from F. subglutinans -like isolates from mango. A second pair of 20-mer primers was developed from a unique fragment in the RAPD assays. It amplified a specific 608 bp fragment for 51 of 54 isolates from mango (all but the three Brazilian isolates). It also amplified a smaller, 550 bp fragment from isolates of F. nygamai (MP-G), but did not amplify DNA of isolates of any other taxon of Fusarium that was tested.     

Population Structure of Phytophthora cinnamomi in South Africa

ABSTRACT Phytophthora cinnamomi isolates collected from 1977 to 1986 and 1991 to 1993 in two regions in South Africa were analyzed using isozymes. A total of 135 isolates was analyzed for 14 enzymes representing 20 putative loci, of which four were polymorphic. This led to the identification of nine different multilocus isozyme genotypes. Both mating types of P. cinnamomi occurred commonly in the Cape region, whereas, predominantly, the A2 mating type occurred in the Mpumalanga region of South Africa. A2 mating type isolates could be resolved into seven multilocus isozyme genotypes, compared with only two multilocus isozyme genotypes for the A1 mating type isolates. Low levels of gene (0.115) and genotypic (2.4%) diversity and a low number of alleles per locus (1.43) were observed for the South African P. cinnamomi population. The genetic distance between the Cape and Mpumalanga P. cinnamomi populations was relatively low (D(m) = 0.165), and no specific pattern in regional distribution of multilocus isozyme genotypes could be observed. The genetic distance between the "old" (isolated between 1977 and 1986) and "new" (isolated between 1991 and 1993) P. cinnamomi populations from the Cape was low (D(m) = 0.164), indicating a stable population over time. Three of the nine multilocus isozyme genotypes were specific to the "old" population, and only one multilocus isozyme genotype was specific to the "new" population. Significant differences in allele frequencies, a high genetic distance (D(m) = 0.581) between the Cape A1 and A2 mating type isolates, significant deviations from Hardy-Weinberg equilibrium, a low overall level of heterozygosity, and a high fixation index (0.71) all indicate that sexual reproduction occurs rarely, if at all, in the South African P. cinnamomi population.     

Variation in susceptibility to pitch canker disease, caused by Fusarium circinatum, in native stands of Pinus muricata

Two hundred Pinus muricata trees, located within a native forest near Monterey, California, were inoculated with the pitch canker pathogen. Treated branches were removed 10–13 weeks following the initial inoculation, and the lengths of the lesions that developed at each of the inoculation sites were measured. Results indicated that bishop pine exhibits a wide range of variation in susceptibility to pitch canker disease. Of the trees that received only one inoculation, 27% showed almost no lesion development, indicating that they were relatively resistant to the pathogen, while others had significantly longer lesions and thus were more susceptible. Clonal propagation and seed collection from resistant individuals may offer useful strategies for disease management in the future.     

Ceratocystis species infecting stem wounds on Eucalyptus grandis in South Africa

Reports of Ceratocystis spp. causing disease of exotic plantation hardwood species have increased in recent years. Ceratocystis fimbriata causes wilt and canker on Eucalyptus spp. in Africa and South America, and C. albofundus results in wilt and death of Acacia mearnsii in Africa. Ceratocystis spp. generally infect wounds on trees, and artificial stem wounding can thus be used to determine the presence of these fungi. The aim of this study was to identify Ceratocystis spp. infecting wounds on Eucalyptus grandis in South Africa. Isolated Ceratocystis spp. were identified using morphological characteristics and comparisons of DNA sequence data for the ITS and 5·8S regions of the rRNA operon. Pathogenicity trials were conducted in the greenhouse to determine the possible role that these Ceratocystis spp. could have in disease development. These trials were also conducted under field conditions. Three Ceratocystis spp. were collected: C. fimbriata , C. moniliformis and C. pirilliformis . This is the first report of C. fimbriata and C. pirilliformis from Eucalyptus spp. in South Africa, and the first report of the latter fungus outside Australia. Both C. fimbriata and C. pirilliformis caused significant lesions on inoculated E. grandis trees. This is the first evidence that C. pirilliformis is a pathogen of Eucalyptus spp. From the results of both greenhouse and field trials, it has the potential to cause serious disease problems in Eucalyptus plantations.     

Comparison of populations of the wilt pathogen Ceratocystis albifundus in South Africa and Uganda

Ceratocystis albifundus is an important fungal pathogen of Acacia mearnsii trees in South Africa. In a previous study, a high level of gene diversity was demonstrated in a South African population of C. albifundus . This, together with the occurrence of the pathogen on native Protea species and its exclusive occurrence in South Africa, led to the hypothesis that C. albifundus is probably native to that country. More recently, C. albifundus has been reported from A. mearnsii in south-western Uganda. The aim of this study was to compare the populations of C. albifundus from Uganda and South Africa based on genetic diversity, population structure and possible gene flow. This was achieved using codominant microsatellite markers developed for the closely related species Ceratocystis fimbriata . Available isolates for comparison were from six different areas of South Africa and six jungle stands in Uganda. Eight of the 11 available markers amplified loci in C. albifundus . Gene diversity was higher in the Ugandan population, but genotypic diversity was greater for the South African isolates. There were no common genotypes between the two populations and they shared only 22% of the total alleles. The populations were genetically isolated from each other and highly substructured within. There was no association between isolates collected from the same geographic locations, and gene flow between the two populations was low. Results suggest that C. albifundus was probably not introduced into Uganda from South Africa but rather that an ancestral population, yet to be discovered, is the source of both populations.     

Relative significance of nursery infections and orchard inoculum in the development and spread of apple canker (Nectria galligena) in young orchards

Three nurseries produced apple rootstocks (M9) and budwood (cv. Royal Gala), which they exchanged at the end of the first year. Each nursery then budded its own budwood onto the rootstocks it had produced and that from the other two nurseries. Budded trees were grown on for a further year before being planted at HRI, East Malling in southern England; NIHPBS, Loughgall in Northern Ireland; and ADAS, Rosemaund in the West Midlands of England. Canker development was monitored twice a year. The position of the infected trees within the orchard was recorded, as was the position of the canker on each tree (main-stem or peripheral). Nectria galligena was isolated from representative cankers and analysed using molecular techniques. At the sites in Northern Ireland and HRI there was a strong positional effect, especially of peripheral cankers, indicating that most of the inoculum was external and had been spread from neighbouring orchards. There was little or no positional effect on main-stem cankers at any of the three sites. The proportions of different isolates taken from peripheral cankers was different in Northern Ireland from that in England, suggesting different populations associated with the geographic areas. In contrast, the populations of N. galligena obtained from main-stem cankers were very similar in England and Northern Ireland. It was concluded that a small proportion of trees developing canker were infected during propagation, with no symptom development until after planting. In a second trial it was demonstrated that trees infected during the propagation phase, and particularly at budding and heading back, could develop canker up to 3 years later. While it is clear that some canker developing in the orchard can be associated with the nursery of production, in climatic conditions conducive to the formation and dissemination of conidia, inoculum from surrounding infected orchards is the primary source of the pathogen. Aerial spread is therefore an essential element of the epidemiology of N. galligena, and its control is a crucial part of any canker-control programme.     

Microsatellite and mating type markers reveal unexpected patterns of genetic diversity in the pine root‐infecting fungus Grosmannia alacris

Grosmannia alacris is a fungus commonly associated with root‐infesting bark beetles occurring on Pinus spp. The fungus has been recorded in South Africa, the USA, France, Portugal and Spain and importantly, has been associated with pine root diseases in South Africa and the USA. Nothing is known regarding the population genetics or origin of G. alacris, although its association with root‐infesting beetles native to Europe suggests that it is an invasive alien in South Africa. In this study, microsatellite markers together with newly developed mating type markers were used to characterize a total of 170 isolates of G. alacris from South Africa and the USA. The results showed that the genotypic diversity of the South African population of G. alacris was very high when compared to the USA populations. Two mating types were also present in South African isolates and the MAT1‐1/MAT1‐2 ratio did not differ from 1:1 (χ² = 1·39, P = 0·24). This suggests that sexual reproduction most probably occurs in the fungus in South Africa, although a sexual state has never been seen in nature. In contrast, the large collection of USA isolates harboured only a single mating type. The results suggest that multiple introductions, followed by random mating, have influenced the population structure in South Africa. In contrast, limited introductions of probably a single mating type (MAT1‐2) may best explain the clonality of USA populations.     

Molecular characterization of Endothia gyrosa isolates from Eucalyptus in South Africa and Australia

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 .