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.     

Association of the pitch canker fungus, Fusarium subglutinans f.sp. pini, with Monterey pine seeds and seedlings in California

The causal agent of pitch canker disease of pines, Fusarium subglutinans f.sp. pini , is a recent introduction to California. The work presented here demonstrates that this pathogen is seed-borne in Monterey pine ( Pinus radiata ). The pathogen was isolated from up to 83% of seeds collected from cones on recently infected branches. Seedling emergence from infested Monterey pine seeds was 9%, compared with 67% for uninfested seeds. The fungus was isolated more frequently from seedlings originating from diseased branches than from symptomless branches. However, more than 50% of seeds from symptomless Monterey pine branches produced symptomless seedlings from which the pathogen was isolated. It is hypothesized that F. s . pini is carried within the seeds, where it may remain dormant until germination. The implications of these findings for the regeneration of native and landscape-planted Monterey pines, and for the transport of this non-native pathogen in pine seeds and seedlings to previously uninfested areas, are discussed.     

Diversity and differentiation in two populations of Gibberella circinata in South Africa

Gibberella circinata [anamorph Fusarium circinatum (=  F. subglutinans f.sp. pini )] causes pitch canker and is an important pathogen in South African pine nurseries. The initial outbreak of the pitch canker fungus was limited to a single nursery at Ngodwana in Mpumalanga Province. Subsequently, several other pine nurseries in South Africa became infected. Most of these outbreaks were relatively small except for the outbreak in the Klipkraal nursery (Mpumalanga Province). The genetic diversity, population differentiation and relative frequencies of the sexual and asexual cycles among two South African subpopulations were determined to establish whether immigration, mutation and/or recombination contributed towards population structure. The allelic diversity of the initial population (Ngodwana) was observed to be lower (0·16) than that of the more recent Klipkraal population (0·25). Approximately 4% ( G _ST = 0·04) of total gene diversity could be attributed to differences among the subpopulations. Furthermore, six new vegetative compatibility groups (VCGs) have been identified since the initial outbreak of G. circinata in South Africa 10 years ago. The relatively low allelic diversity and low level of genetic differentiation suggest restricted gene flow among subpopulations, and indicate that the pathogen has been introduced recently. However, the amount of allelic and VCG diversity suggests that multiple genotypes have been introduced into South Africa. The increases in effective population number, allelic diversity and new VCGs over the past 10 years suggest that sexual reproduction might be occurring.     

Variation in rates of spore deposition of Fusarium circinatum, the causal agent of pine pitch canker, over a 12-month-period at two locations in Northern California

Patterns of spore deposition by Fusarium circinatum, the causal agent of pine pitch canker (PPC) of Monterey pine (Pinus radiata) and other conifers, were studied between May 2003 and April 2004 at two sites in Northern California using a novel spore trapping method combined with a real-time polymerase chain reaction (PCR) approach. At each study site, two plots were sampled by placing spore traps at 100 m intervals along transects 600 m in length. The air was sampled continuously by exchanging the spore traps every 2 weeks. The spore deposition rate (DR), ranged from 0 to 1.3 x 10(5) spores m(2). Spores were detected throughout the year, with higher trapping frequencies (TF) during the rainy season (November to April), than during the dry season (May to October). The detection of spores on traps at distances larger than 200 m from any Monterey pine, suggests at least midrange aerial dispersal. Finally, different inoculum loads were associated with trees displaying different levels of disease symptoms, suggesting infectiousness of the pathogen varies as the disease progresses. This study represents one of the first documenting continuous inoculum pressure values over an entire year for a forest pathogen, and provides important epidemiological information that will be invaluable in the development of disease progression models.     

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.     

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.     

Temporal variation in contamination of pine engraver beetles with Fusarium circinatum in native Monterey pine forests in California

The relative importance of beetle species associated with Fusarium circinatum‐infected Monterey pines was investigated in three Monterey pine forests along the coast of central California, USA from April to November in 2004 and 2005. Fusarium circinatum was frequently isolated from Ips mexicanus and I. plastographus. The mean percentage isolation based upon numbers of I. mexicanus and I. plastographus carrying propagules of F. circinatum was 17·7 and 10·9% in 2004 and 16·7 and 17·3% in 2005, respectively. The mean percentage isolation was high in the spring and early summer and low in late summer and autumn in all three locations for both species. Isolation was higher from beetles emerging from harvested F. circinatum‐infected pine‐stems than for trapped beetles, 42·4% for I. mexicanus and 45·9% for I. plastographus. The mean (± SE) propagule load of trapped I. mexicanus was 269·5 (± 14·1) in 2004 and 281·7 (± 35·7) in 2005 and was 216·1 (± 28·9) in 2004 and 251·9 (± 28·4) in 2005 for I. plastographus. Mean propagule loads decreased from May to November in all locations for both species. Propagule loads of beetles emerged from infected stems were lower than that of trapped beetles, with means of 89·4 (± 23·2) and 93·0 (± 23·2) for I. mexicanus and I. plastographus, respectively. Thus beetles must acquire fungal propagules from more than one infected host. These results also suggest that higher contamination rates and propagule loads in spring and early summer may indicate a higher risk of pitch canker transmission, relative to late summer or autumn.     

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.     

Susceptibility of pines in South Africa to the pitch canker fungus subglutinans f.sp. pini

Fusarium subglutinans f.sp. pini (F.s. pini)is the causal agent of pitch canker of pines. The fungus has recently been found in South Africa on the diseased roots of seedlings, but has as yet not been detected on mature trees in commercial forests. Inoculation of 1 -year-old and seedlings with isolates of resulted in canker development and shoot mortality. No significant differences in virulence were found among eight isolates of the pathogen on and but isolate MRC 6214 was significantly more virulent on seedlings than MRC 6209. Disease development was significantly more severe on and than on Pathogenicity tests on 4-year-old and trees yielded comparable results. Resinous cankers, similar to those described for pitch canker, developed on trees in the vicinity of inoculation points but development ceased before stems were girdled.     

Influence of host resistance on the genetic structure of the white pine blister rust fungus in the western United States

Cronartium ribicola, the causal agent of white pine blister rust, has been devastating to five-needled white pines in North America since its introduction nearly a century ago. However, dynamic and complex interactions occur among C. ribicola, five-needled white pines, and the environment. To examine potential evolutionary influences on genetic structure and diversity of C. ribicola in western United States, population genetic analyses of C. ribicola were conducted using amplified fragment length polymorphism (AFLP) molecular markers. The fungus was sampled at six sites. Collections for two of the six sites were from separate plantings of resistant-selected western white pine and sugar pine. Heterozygosity based on polymorphic loci among populations ranged from 0.28 to 0.40, with resistant-selected plantations at the extremes. Genetic differentiation was also highest between these two populations. Principal coordinates analysis and Bayesian assignment placed most isolates that are putative carriers of virulence to major-gene resistance into a discernable cluster, while other isolates showed no clustering by site or host species. These results indicate that C. ribicola in western North America is not genetically uniform, despite its presumed single site of introduction and relatively brief residence. Moreover, major-gene resistance appears to have imposed strong selection on the rust, resulting in reduced genetic diversity. In contrast, no evidence of selection was observed in C. ribicola from hosts that exhibit only multigenic resistance.     

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.     

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.     

A Rapid Diagnostic Test to Distinguish Between American and European Populations of Phytophthora ramorum

ABSTRACT A new devastating disease in the United States, commonly known as Sudden Oak Death, is caused by Phytophthora ramorum. This pathogen, which previously was described attacking species of Rhododendron and Viburnum in Germany and the Netherlands, has established itself in forests on the central coast of California and is killing scores of native oak trees (Lithocarpus densiflora, Quercus agrifolia, Q. kelloggii, and Q. parvula var. shrevei). The phytosanitary authorities in the European Union consider non-European isolates of P. ramorum as a threat to forest trees in Europe. To date, almost all European isolates are mating type A1 while those from California and Oregon are type A2. The occurrence of both mating types in the same region could lead to a population capable of sexual recombination, which could generate a new source of diversity. To prevent contact between these two populations, a rapid, reliable, and discriminating diagnostic test was developed to easily distinguish the two populations. Based on a DNA sequence difference in the mitochondrial Cytochrome c oxidase subunit 1 (Cox1) gene, we developed a single-nucleotide polymorphism (SNP) protocol to distinguish between isolates of P. ramorum originating in Europe and those originating in the United States. A total of 83 isolates of P. ramorum from Europe and 51 isolates from the United States were screened and all isolates could be consistently and correctly allocated to either the European or the U.S. populations using the SNP protocol.     

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.     

马尾松(Pinus massoniana)感染松材线虫(Bursaphelenchus xylophilus)的研究

 1987-1988年在江苏南京-镇江松材线虫疫区内采(征)集143份自然枯死或濒死马尾松样本,在94份(65.7%)样本中查见松材线虫。有关林分中马尾松的病枯情况不一,在句容桥头一下蜀一线、江宁汤山和六合方山等处有过相当多的马尾松发病死亡。表明松材线虫是当地马尾松自然枯死的主要原因。人工测定了无黑松林区5年生浙江富阳马尾松苗及混栽黑松林区4-5年生江苏马尾松苗对两种松材线虫分离物的反应,结果指出,供试松苗都表现中度感染或高度感染,并有20%或70-80%的病枯株。接种林间马尾松,初步证实马尾松对松材线虫的抗性随树龄变化。1-2年生马尾松苗高感松材线虫,病枯株率高达85.7%;树龄25年以上的成材树病枯株率约30%;而10年左右的幼树则无一发病死亡。     

Impacts of exotic forest pathogens on Mediterranean ecosystems: four case studies

Mediterranean ecosystems are hotspots of biodiversity. Because of a coincidence of high species richness and human presence, Mediterranean biodiversity is particularly threatened by processes such as habitat degradation, fragmentation and loss, pollution, climate change and introduction of invasive species. Invasive tree pathogens are among the problematic exotic species of California, Chile, the Mediterranean, South Africa and Australia. In this review, we provide an update on a selection of non-native tree pathogens currently posing a threat in Mediterranean ecosystems. The impact of exotic forest pathogens range from large-scale tree and shrub mortality in native ecosystems (Phytophthora ramorum on the West Coast of the USA) to disruption of plantations of exotic (e.g., Seiridium cardinale on planted Monterey cypress in California, Fusarium circinatum on Monterey pine worldwide) and native trees (introduction of the North American Heterobasidion irregulare in stone pine woodland in Italy). Genetic analyses are instrumental in improving our understanding and management of these outbreaks. There is a need for more empirical data on how novel pathosystems are likely to develop under novel climates, as well as interdisciplinary collaborations among forest pathologists, theoretical modellers and climatologists. The magnitude of the observed effects of some exotic tree diseases makes it important to try and minimize the risk of the inadvertent movement of plant pathogens when planning assisted migration activities to enable plant species to cope with rapid climate change.     

Plasticity in plant-microbe interactions: a perspective based on the pitch canker pathosystem

What we know about the life history of fungi that cause disease in plants is commonly based on studies of the pathogen’s interaction with a susceptible host: how and when infection occurs, growth and reproduction within the host, and survival during the interval when a growing host is not available. This focus is appropriate, given the need for information that will facilitate management of disease affecting an economically important crop, but it can limit recognition of the full range of resources that may be utilized by fungi that we classify as plant pathogens. This was certainly the case for Fusarium circinatum, which causes a destructive disease of pines known as pitch canker. Although F. circinatum was initially known only as a necrotrophic, wound-infecting pathogen of coniferous trees, recent research has revealed that an isolate of this fungus that will kill shoot tissue when inoculated into a wound can also have a biotrophic relationship with roots of pine seedlings, infect and grow within grasses without causing symptoms, and cause ear rot of corn. Thus, although F. circinatum became known to science because it induced visible symptoms on pines, it has the capacity for a much broader range of ecological activities than is captured by its designation as a necrotrophic pathogen. The physiological plasticity manifested by F. circinatum illustrates the challenge of obtaining a comprehensive understanding of the life history of a plant pathogenic fungus.     

Vegetative Compatibility Groups in Verticillium dahliae Isolates from the Netherlands as Compared to VCG Diversity in Europe and in the USA

In a study of vegetative compatibility in Verticillium dahliae in the Netherlands, a collection of 45 isolates including representatives from woody hosts, several horticultural crops and from the soil of potato fields was examined. In addition an effort was made to compare vegetative compatibility groups (VCGs) from different countries. The results of this study indicate that VCG diversity in V. dahliae in the Netherlands is limited. Only two VCGs were detected: VCG NL-I and VCG NL-II. The former is the predominant VCG for isolates from tree hosts. However, Verticillium wilt in trees can be caused by isolates from both VCGs. It is suggested that the predominance of VCG NL-I in tree hosts is the result of the origin of the tree and the cropping history of its growing site, rather than trees being preferential hosts for isolates from this VCG. Comparison of VCG testers from the Netherlands, from several other European countries and from the USA show that in Europe two major VCGs are present. The first one, including NL-I, is compatible with USA VCG 3 and VCG 4, whereas the second one, including NL-II, is compatible with USA VCG 1 and VCG 2. These groups are not completely separated; in some cases, testers formed heterokaryons with VCG testers from both main groups. Because of the presence of these bridge isolates and because mutants from the same isolate differ in ability to form heterokaryons, it is emphasised that careful selection of isolate testers is an essential step to get a clear picture of VCG diversity.     

Evidence of Cytoplasmic Inheritance of Virulence in Cronartium ribicola to Major Gene Resistance in Sugar Pine

ABSTRACT Tests for Mendelian segregation of virulence and avirulence in Cronartium ribicola, causal agent of white pine blister rust, to a major gene (R) for resistance in sugar pine were made using haploid basidiospore progenies from single diploid telia as inoculum on resistant genotypes. The telia were sampled from a small deme in the Siskyou Mountains of northern California, where a few mature sugar pines known to be Rr genotypes had become infected after withstanding the chronic blister rust epidemic for several decades and where intermediate frequencies of virulence in the ambient basidiospore population were subsequently measured. Infection type on inoculated seedlings with R was qualitative: all progenies of 81 single telia tested over 3 different years were either virulent (compatible) or avirulent (inducing hypersensitive necrosis), never a mixture of both reactions. The complete absence of heterozygotes in the telia population is strong evidence that virulence is not controlled by a nuclear gene. The data are consistent with earlier tests showing that basidiospore inoculum derived from aeciospores isolated from infected Rr trees produced mostly (>90%) virulent reactions on R- seedlings. The evidence indicates that transmission of virulence is uniparental via the cytoplasm of aeciospores. Exchange of spermatia between haploid thalli does not appear to be involved.     

Molecular Variability Within and Among Verticillium dahliae Vegetative Compatibility Groups Determined by Fluorescent Amplified Fragment Length Polymorphism and Polymerase Chain Reaction Markers

ABSTRACT A degree of genetic diversity may exist among Verticillium dahliae isolates within vegetative compatibility groups (VCGs) that bears phytopathological significance and is worth investigating using molecular tools of a higher resolution than VCG characterization. The molecular variability within and among V. dahliae VCGs was studied using 53 artichoke isolates from eastern-central Spain, 96 isolates from cotton, 7 from cotton soil, and 45 from olive trees in countries of the Mediterranean Basin. Isolates were selected to represent the widest available diversity in cotton- and olive-defoliating (D) and -nondefoliating (ND) pathotypes, as well as for VCG. The VCG of 96 cotton and olive isolates was determined in this present study. Molecular variability among V. dahliae isolates was assessed by fluorescent amplified fragment length polymorphism (AFLP) analysis and by polymerase chain reaction (PCR) assays for DNA fragments associated with the D (462 bp) and ND (824 bp) pathotypes, as well as a 334-bp amplicon associated with D pathotype isolates but also present in some VCG2B isolates. Isolates from cotton were in VCG1A, VCG1B, VCG2A, VCG2B, and VCG4B and those from olive trees were in VCG1A, VCG2A, and VCG4B. Artichoke isolates included representatives of VCG1A, VCG2A, VCG2B (including a newly identified VCG2Ba), and VCG4B. AFLP data were used to generate matrixes of genetic distance among isolates for cluster analysis using the neighbor-joining method and for analysis of molecular variance. Results demonstrated that V. dahliae isolates within a VCG subgroup are molecularly similar, to the extent that clustering of isolates correlated with VCG subgroups regardless of the host source and geographic origin. VCGs differed in molecular variability, with the variability being highest in VCG2B and VCG2A. For some AFLP/VCG subgroup clusterings, V. dahliae isolates from artichoke grouped in subclusters clearly distinct from those comprising isolates from cotton and olive trees. In addition, VCG2B isolates from artichoke formed two distinct clusters that correlated with PCR markers of 334 bp (VCG2B(334)) or 824 bp (VCG2B(824)). Artichoke isolates in the VCG2B(334)/2beta(334) cluster were molecularly similar to isolates of VCG1A. The molecular difference found among artichoke isolates in VCG2B correlates with virulence of isolates to artichoke and cotton cultivars demonstrated in a previous study.