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Wounding, stress, virulence and host susceptibility
For many years, it was assumed that D. pinea, especially the B morphotype, required wounds for infection (Wang et al. 1985; Palmer et al. 1987). These wounds would typically originate during pruning, hail damage or through insect feeding (Laughton 1937; Gilmour 1964; Lückhoff 1964; Marks & Minko 1969; Wright & Marks 1970; Peterson 1977; Swart et al. 1985; Zwolinski et al. 1995). This view has changed with various reports of both morphotypes of the fungus being able to infect unwounded stems and leaves through direct penetration of the stomatal pits (Waterman 1943; Brookhouser & Peterson 1971; Palmer 1991; Blodgett & Stanosz 1997a). Diplodia pinea has also been shown to persist in healthy, asymptomatic host tissue and mature, unopened seed cones in a latent state (Smith et al. 1996; Stanosz et al. 1997; Flowers et al. 2001, 2003). Stanosz et al. 1997 demonstrated that both the A and B morphotypes of D. pinea isolated from asymptomatic shoots of P. resinosa and P. banksiana were able to develop characteristic Diplodia die-back symptoms when artificially inoculated. Diplodia pinea sensu lato, like most species of the Botryosphaeriaceae, occur as latent infections in healthy tissue (Slippers & Wingfield 2007). One of the major obstacles in dealing with these latent infections is the lack of ability to detect them easily and to distinguish them from other epiphytic infections. Various quick assays have therefore, been developed to detect latent infections directly from asymptomatic host tissue. Flowers et al. (2003) developed a nested PCR using nuclear rDNA ITS primers to detect the presence of latent D. pinea and B. obtusa infections in pine tissue but without being able to differentiate between these closely related species and the morphotypes of D. pinea. This was followed by a Real-time quantitative PCR assay based on the small ribosomal subunit able to rapidly detect and quantify D. pinea infections in inoculated P. nigra Arnold shoots (Luchi et al. 2005), as well as asymptomatic P. nigra shoots (Maresi et al. 2007). These assays were also unable to distinguish between the morphotypes of the fungus. A species-specific PCR assay, based on polymorphisms in the mitochondrial small subunit ribosome gene (mtSSU rDNA), has since been developed that is able to differentiate between the A and B morphotypes of D. pinea and D. seriata directly from dead red and jack pine tissue (Smith & Stanosz 2006).
Latent D. pinea sensu lato infections are hypothesized to be a survival mechanism of the fungus awaiting opportunity to overcome host defense responses and cause visible disease symptoms (Stanosz et al. 1997; Flowers et al. 2006). The onset of host stress as a result of adverse environmental or physical factors initiates pathogenic D. pinea infections (Laughton 1937; Waterman 1943; Buchanan 1967; Minko & Marks 1973; Brown et al. 1981; Swart et al. 1987a; Stanosz et al. 2001). Hail, drought, overstocking, poor site conditions and nutrient deficiencies are general predisposing factors (Laughton 1937; Lückhoff 1964; Wright & Marks 1970; Minko & Marks 1973; Chou 1977, 1982; Bega et al. 1978; Brown et al. 1981; Bachi & Peterson 1985; Stanosz et al. 2001). Maresi et al. (2007) demonstrated how water stress can potentially be a trigger that enables the fungus to switch from a latent phase to that of a more active pathogenic phase when they found a positive correlation between the presence of D. pinea and the normalized insolation index. The normalized insolation index is a measure of the amount of heat at a point that ultimately is an indication of water stress at a specific site. These predisposing factors decrease the rate of the host defense responses and consequently the growth of the pathogen increases as a result of a larger carbohydrate pool available to it (Schoeneweiss 1981; Bachi & Peterson 1985).
Hail damage followed by D. pinea-induced die-back is a serious problem in South Africa resulting in huge economic losses (Zwolinski et al. 1990a, 1990b). The highest degree of mortality occurs four months after a hailstorm and can last for up to a year, after which regeneration of foliage normally occurs (Zwolinski et al. 1990b). Smith et al. (2002a) mapped the colonization of D. pinea in hail-damaged P. patula trees from the cone pith, through the stipe (connection between the cone and the branch), the branch and finally into the branch pith. In undamaged P. patula trees no discoloration due to D. pinea was found in the branch pith but D. pinea was present latently in the cone pith and in the stem.
Insects are commonly associated with D. pinea infection. They play a role in facilitating the colonization of healthy cambial tissue and thus, enhance the severity and impact of the infection rather than playing a role in the dissemination of the fungus (Haddow & Newman 1942; Wingfield & Palmer 1983; Zwolinski et al. 1995). Examples of insects that have been associated with D. pinea are the pine spittle bug (Aphrophora parallela Say) (Haddow & Newman 1942; Waterman 1943), the pitch nodule moth (Petrova albicapitana Busk) (Hunt 1969), the deodar weevil (Pissodes nemorensis Germar), the bark beetle (Orthomicus erosus Woll.) (Wingfield & Palmer 1983; Zwolinski et al., 1995), the cone bug (Gastrodes grossipes De Geer) (Feci et al. 2002) and the pine shoot moth (Dioryctria sp.) (Feci et al. 2003). Zwolinski et al. (1995) made interesting observations regarding the association of P. nemorensis and O. erosus with post-hail associated Diplodia infections in South Africa. Orthomicus erosus is found only on post-hail Diplodia-infected trees while P. nemorensis can occur on healthy trees but exacerbates the spread of the fungus in post-hail Diplodia-infected trees.
POPULATION GENETICS
The genetic structure of D. pinea sensu lato populations is relevant to the management and quarantine of Diplodia die-back and other Diplodia-associated diseases. A pathogen population with a highly diverse genetic structure can more easily adapt and overcome resistance. Because D. pinea is an endophyte (Smith et al. 1996; Stanosz et al. 1997; Burgess et al. 2001a; Flowers et al. 2001) and found on pine seed collected from cones in seed orchards (Peterson 1977; Fraedrich & Miller 1995; Vujanovic et al. 2000), it is fair to assume that wherever pine seed or seedlings have been introduced the fungus is likely to have been introduced with it. In a study conducted by Smith et al. (2000), genotypic diversity of two D. pinea populations was assessed using vegetative compatibility groups (VCGs). They found the genotypic diversity of an introduced South African population to be unexpectedly higher than that of a native Indonesian population (Smith et al. 2000). In a subsequent study, simple sequence repeats (SSRs), were used to determine the genotypic diversity of four Diplodia populations from native and introduced P. radiata (Burgess et al. 2001a). The same trend was observed with higher genotypic diversities for the introduced South African, New Zealand and Australian populations, with those of South Africa being the highest, followed by New Zealand and Australia compared to a native Californian population (Burgess et al. 2001a). With D. pinea being an asexually reproducing fungus, populations would be expected to be almost clonal with very low genotypic diversities. In the absence of sexual recombination and selective pressure, the assumption was made that the observed genotypic diversity reflects the number of introductions of the fungus into a region (Burgess et al. 2001a; Burgess & Wingfield 2002). Therefore, the high genotypic diversity observed for the introduced D. pinea populations is accounted for by multiple introductions of the fungus together with pine seed into the southern hemisphere (Smith et al. 2000; Burgess et al. 2001a; Burgess & Wingfield 2002). The much higher genotypic diversity calculated for the South African population was linked to the fact that afforestation in South African started about 100 years before Australia and New Zealand and that there has been little control on the importation of seed into the country (Burgess et al. 2001a; Burgess & Wingfield 2002). In contrast, Australia and New Zealand have strict quarantine regulations that significantly restrict the introduction of pine seed into those countries (Burgess et al. 2001a; Burgess & Wingfield 2002).
In the previous two studies, the genetic diversity of D. pinea populations were determined but the existence of D. pinea sensu lato as representing two different morphotypes and potential cryptic speciation were not considered. As previously discussed, the morphotypes of D. pinea differ with regards to their taxonomy, biology and virulence. Populations of the two morphotypes also have different genetic structures. The A morphotype or D. pinea sensu stricto is the main species associated with most Pinus spp. outside their native range (Burgess et al.2004a). While the B morphotype is almost exclusively associated with P. radiata in its native range (Burgess et al. 2004b). The D. pinea sensu stricto populations have very low gene diversities, little population differentiation and share multilocus genotypes between populations on different continents (Burgess et al. 2004a). This suggests a long asexual history and constant selection pressure as selection is linked to the success of the endophyte. D. pinea sensu stricto populations are thus highly unlikely to overcome host resistance and breeding for resistance in the host will be a durable option. In contrast, populations of the B morphotype have high allelic diversity and no multilocus genotypes are shared between populations. The huge genetic distance between populations with limited gene flow suggests a recent history of recombination and/or mutation as well as the presence of a cryptic sexual stage (Burgess et al. 2004b).
CHAPTER 1 Diplodia pinea sensu lato as part of the Botryosphaeriaceae and associated mycoviruses
1. Introduction
2. Taxonomy of the Diplodia pinea species complex
3. Pathogen biology
4. Population genetics
5. Disease management
6. Conclusions
7. References
8. Figures
CHAPTER 2 Multiple gene genealogies and microsatellite markers reflect relationships between the morphotypes of Sphaeropsis sapinea and identify a new species of Diplodia
-Abstract
-Introduction
-Materials and Methods
-Fungal isolates
-DNA extractions
-Amplification of partial protein-coding genes and microsatelite loci
-Sequencing
-Phylogenetic analyses
-Results
-Amplification and sequencing protein-coding genes and microsatelite loci
-Phylogenetic analyses
-Taxonomy
-Discussion
-References
-Table
-Figures
CHAPTER 3 Molecular and morphological characterization of Dothiorella casuarini sp. nov. and other Botryosphaeriaceae with Diplodia-like conidia
-Abstract
-Introduction
-Materials and Methods
-Fungal isolates and morphological characterization
-DNA extractions
-DNA amplification and sequencing
-Phylogenetic analyses
-Results
-Phylogenetic analyses
-Taxonomy
-Discussion
-References
-Table
-Figures
CHAPTER 4 Phylogeny of the Botryosphaeriaceae reveals patterns of host association
-Abstract
-Introduction
-Materials and Methods
-Fungal isolates
-DNA extractions, amplification and sequencing
-Phylogenetic analyses
-Results
-Phylogenetic analyses of Botryosphaeriaceae with Diplodia-like anamorphs
-Phylogenetic analyses for seven lineages in Botryosphaeriaceae
-Discussion
-References
-Tables
-Figures
CHAPTER 5 Patterns of multiple virus infections in the conifer pathogenic fungi, Diplodia pinea and D
scrobiculata.
-Abstract
-Introduction
-Materials and Methods
-DsRNA extraction, cDNA synthesis and cloning of a putative RdRp gene
-Primer development
-Fungal isolates used for genotyping
-Total RNA isolations
-cDNA synthesis and Real-time PCR genotyping
-Amplicon sequence confirmation
-Results
-Partial characterization of a putative RdRp gene
-cDNA synthesis and Real-time PCR genotyping
-Amplicon sequence confirmation
-Virus distribution in isolates
-Discussion
-References
-Table
CHAPTER 6 Characterization of a novel dsRNA element associated with the pine endophytic fungus, Diplodia scrobiculata
-Abstract
-Introduction
-Materials and Methods
-Fungal isolate and dsRNA extraction
-Synthesis and cloning of cDNA using random hexamer primers
-Amplification and cloning of the complete viral genome
-Determination of the distal ends of the viral genome
-Isolation and amplification of genomic DNA
-Sequencing and sequence analysis
-Phylogenetic analysis
-Results
-Synthesis and sequencing of cDNA from D. scrobiculata dsRNA
-Genome organization of DsRV1
-Amplification of genomic DNA
-Phylogenetic analysis
-Discussion
-References
-Table
-Figures
SUMMARY
