Relative pathogenicity of Cryphonectria cubensis on Eucalyptus clones differing in their tolerance to C. cubensis

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Biology & Symptoms:

Cryphonectria canker is characterised by sunken elongated areas at the bases or higher up on infected trees (Fig. 1A). The tissue below the bark is typically brown and dead, with kino exudation usually observed on older cankers (Fig. 1B) (Boerboom & Maas 1970; Sharma et al. 1985). In South Africa, only basal cankers have been observed, which is different to the situation in other parts of the world where cankers are commonly found higher up in trees (Hodges et al. 1979; Sharma et al. 1985; Wingfield et al. 1989). Trees react to C. cubensis infection by producing callus around the site of invasion (Hodges et al. 1979). Cryphonectria canker tends to be more severe on actively growing trees. Thus, the development of cankers is limited by stress factors such as drought, which results in smaller cambial lesions (Swart et al. 1992). This is consistent with the epidemiology of the disease that is known to occur predominantly in higher rainfall areas in South Africa and elsewhere in the world (Hodges et al. 1979; Sharma et al. 1985; Florence et al. 1986; Wingfield et al. 1989). Rainfall (2000-2400 mm/ annum) and temperatures above 23ºC are known to favour Cryphonectria canker (Sharma et al. 1985; Florence et al. 1986).

HYPOVIRUS

Historical overview: Cryphonectria hypovirus 1 and the Cryphonectria hypovirus 2 are the only species in the genus Hypovirus, with two other tentative species, Cryphonectria hypovirus 3/GH2 and the Cryphonectria hypovirus 4/SR2 (Hillman et al. 2000a). These Cryphonectria hypoviruses all infect C. parasitica, the causal agent of chestnut blight. Cryphonectria parasitica was first reported in 1904 in North America where it has been responsible for the devastation of the American chestnut (Castanea dentata Borkh.) (Merkel 1906). In 1938, this disease appeared in Italy in the province of Genoa on the European chestnut Castanea sativa Mill. (Pavari 1949). However, Biraghi (1950) observed the spontaneous healing of cankers on sprouts growing from the stem of a chestnut tree. Studying this phenomenon led to the isolation of hypovirulent strains of C. parasitica (Grente 1965). Subsequently, the factor causing hypovirulence was shown to be transmissible via hyphal anastomosis.
Fungal isolates harbouring this factor had the ability to heal actively growing cankers on trees after inoculation (Grente & Sauret 1969; Grente & Berthelay-Sauret 1978). Healing blighted trees were also observed in North America in 1976, which led to the isolation of a hypovirulent isolate in that country (Anagnostakis 1982a). A detailed study of C. parasitica strains from both North America and Europe has shown that hypovirulence is consistently associated with dsRNA infections (Day et al. 1977). The molecular weights and the concentration of the dsRNA in isolates from North America and Europe differed, distinctly. The molecular weights were between 4.0 and 7.0 x 106 and the concentrations of dsRNA were lower in the American than the European isolates (Dodds 1980). Dot blot hybridisation has confirmed the lack of sequence homology between the ds RNA elements of the European and the American strains of C. parasitica (L’Hostis et al. 1985). Curing these hypovirulent isolates using cycloheximide, resulted in an increase in the virulence converting the effect of the dsRNA on virulence (Fulbright 1984).

Transmission of dsRNA:

For the effective application of mycoviruses in any biological control system, it is important to have a clear understanding regarding the transmission of dsRNA, through the fungal population. DsRNAs are known to be transmitted by two means in fungal populations. This is either vertically to a different level through spores or horizontally via hyphal anastomosis (Nuss 1996). The movement of dsRNA via hyphal anastomosis is favoured when isolates belong to the same vegetative compatibility group (Anagnostakis 1977). University of Pretoria etd – Van Heerden, S W (2004) 11 Garbelotto et al. (1992) showed that the Italian C. parasitica population consists of a few vegetative compatibility groups (VCGs). Thus, by using five hypovirulent strains, they were able to convert 77% of the isolates to the hypovirulent phenotype. In North America the C. parasitica population has a much higher level of genetic diversity than it has in Europe, which might explain the unsuccessful dissemination of hypovirulence in North America (Anagnostakis 1982a; Anagnostakis et al. 1986; Anagnostakis 1987; Heiniger & Rigling 1994).
In C. parasitica, vegetative incompatibility is controlled by six vegetative incompatibility (vic) loci, each with two alleles (Anagnostakis 1982b; Cortesi & Milgroom 1998). Liu & Milgroom (1996) have further shown that a negative correlation exists between hypovirus transmission and the number of vic genes that differ between isolates of C. parasitica. Thus, the level of hypovirus transmission will decrease as the number of vic genes, differing between the donor and recipient isolate, increases (Liu & Milgroom 1996). Hypovirus transmission can still occur between fungal strains that are unable to form heterokaryons, due to different alleles at a single vic locus (Huber & Fulbright 1994; 1995). It was later concluded that the transmission of viruses in C. parasitica is primarily controlled by the vic genes (Cortesi et al. 2001)

Genetic engineering of fungal hypoviruses

The completion of the full genome sequence of the C. parasitica hypovirus CHV1-EP713 (Shapira et al. 1991), allowed for the construction of a full-length cDNA clone of this virus (Choi & Nuss 1992b). Choi and Nuss (1992b) used this full-length cDNA for transformation into a virus-free C. parasitica strain. Transformants had the hypovirulence phenotype and they also contained a chromosomally integrated copy of the virus as well as a cytoplasmically replicating form (Choi & Nuss 1992b). As mentioned earlier in this review, hypoviruses are not transmitted to the ascospore progeny and the transmission into conidia does not occur consistently (Nuss 1996). The construction of an infectious cDNA copy of CHV1-713 has overcome these problems. Chen et al. (1993) have used repeated rounds of conidiation to show that the chromosomally integrated viral cDNA copy is stable and that the virus can be transmitted to ascospore progenies. This is a novel form of transmission, since the progenies contain a range of different VC groups due to allelic rearrangement at the vic loci (Nuss et al. 2002). The transgenenic strains, therefore, have enhanced dissemination properties and thus enhanced biological control properties. A reporter gene was also incorporated into University of Pretoria etd – Van Heerden, S W (2004) 13 Cryphonectria transgenic strains.
For this purpose the green fluorescent protein (GFP) gene from Aequorea victoria was used to track the movement of hypoviruses through hyphal anastomosis from strain to strain (Suzuki et al. 2000). The transfection of spheroplasts produced from virus free C. parasitica isolates with the full length in vitro produced CHV1-EP713 transcripts, using electroporation has been successful (Chen et al. 1994). In this case, the success of the transfection protocol relies on hyphal anastomosis. After electroporation, the spheroplasts are plated onto a regeneration medium and the RNA present in a small number of successfully transfected speroplasts, will spread through the colony (Nuss et al. 2002). This transfection strategy has made it possible to expand the range of fungi that can be infected by CHV1-EP713. Three species in the genus Cryphonectria namely C. cubensis, C. havanenesis (Bruner) Barr and C. radicalis (Schw.:Fries) Barr, and one species in the genus Endothia namely E. gyrosa (Schw.: Fries) Fries have been successfully transfected with the C. parasitca hypovirus RNA (Chen et al. 1994). These transfections resulted in phenotypic changes in the recipient fungi. The phenotypic changes observed for C. radicalis were similar to those of hypovirulent C. parasitica isolates, including reduced growth rate, reduced sporulation and a suppression of the orange pigmentation (Chen et al. 1994). Further, Chen et al. (1994) showed that transfected E. gyrosa and C. cubensis isolates had increased bright orange pigment, whereas transfected C. havanensis had only slight morphological change. In addition, a study by Chen et al. (1996) showed that virus transmission to the asexual spores ranges from 0% for C. cubensis to 50-100% for C. parasitica.

Isolate selection and growth studies

One hundred bark samples from trees infected with C. cubensis were randomly collected in KwaZulu-Natal, South Africa. The fungus was induced to sporulate and isolations were made using the method previously described by van Heerden & Wingfield (2001). All isolates used in this study are maintained in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria. One hundred C. cubensis isolates were inoculated onto 2% Malt extract agar (MEA) and incubated at 25ºC for 7 days at normal illumination cycles (12 hours light and 12 hours darkness). A 5 mm diam. mycelial plug from each of these isolates was inoculated in the middle of two 9 cm diam. Petri dishes containing 2% MEA. The plates were incubated at 25ºC. Colony diameters were measured four days after inoculation. Seven slow growing (CMW11325, CMW11332, CMW11341, CMW11353, CMW11354, CMW11342 and CMW11329) and two rapidly growing (CMW11355 and CMW11351) South African C. cubensis isolates were selected for dsRNA extraction (Table 1). These isolates were grown in Erlenmeyer flasks containing 2% Malt extract broth at 25°C. The mycelium was harvested after sufficient growth, freeze-dried and stored at -20°C before use.

Extraction and purification of dsRNA

Double stranded (ds) RNA was extracted using the method of Valverde et al. (1990) with modifications outlined by Preisig et al. (1998). One gram lyophilised ground mycelium was transferred to 40 ml centrifuge tubes into which 10 ml 2 x STE (0.2 M NaCl, 0.1 M Tris HCl, 2 mM EDTA pH 8, pH 6.8) and 1% SDS was added. Samples were mixed using a vortex mixer and subsequently incubated at 60°C for 10 min. After incubation, 10 ml Phenol (pH 7.5) was added. The solution was shaken at room temperature for 30 min and centrifuged in a Beckman JA25.50 rotor for 30 min at 15000 rpm. The aqueous phase was transferred to clean centrifuge tubes and 10 ml of chloroform was added. The samples were mixed with a vortex mixer before being centrifuged at 10000 rpm for 15 min. This step was repeated until the inter phase was free of protein. After the last extraction step, the aqueous phase was transferred to a clean centrifuge tube and 16% absolute ethanol was added and centrifuged at 5000 rpm for 5 min to remove the chromosomal DNA.

Table of Contents :

• Acknowledgements
• Preface
• Chapter 1:
  • Cryphonectria cubensis in South Africa, and opportunities for biological control via
  • hypovirulence: A review
  • 1. Introduction:
  • 2. Taxonomy and Biology of Cryphonectria cubensis
  • 2.1 Geographical distribution and origin
  • 2.2 Host range
  • 2.3 Biology and symptoms
  • 2.4 Control strategies and factors influencing their efficacy
  • 3. Hypovirulence in Fungi
  • 3.1 Introduction
  • 3.2 Hypovirus
    • 3.2.1 Historical overview
    • 3.2.2 Phenotypic changes associated with the presence of dsRNA
    • 3.2.3 Genome organisation and structure
    • 3.2.4 Transmission of dsRNA
    • 3.2.5 Application of biological control
    • 3.2.6 Genetic engineering fungal hypoviruses
  • 3.3 Mitovirus
  • 4. Conclusion
• Chapter 2:
  • Molecular characterisation of mitoviruses co-infecting South African isolates of the Eucalyptus canker pathogen Cryphonectria cubensis
  • University of Pretoria etd – Van Heerden, S W (2004)
• Chapter 3:
  • Relative pathogenicity of Cryphonectria cubensis on Eucalyptus clones differing in their tolerance to C. cubensis
• Chapter 4:
  • Transfection studies with the Diaporthe RNA virus (DaRV) and other Cryphonectria cubensis isolates
• Chapter 5:
  • Biological control of Cryphonectria canker of Eucalyptus using an isolate transfected with the C. parasitica hypovirus
  • Summary

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Studies on Cryphonectria cubensis in South Africa with special reference to mycovirus infection