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DNA profiling can help to identify which Mycosphaerella species are attacking your blue gums

Brad Potts
University of Tasmania

In evaluating genetic differences in susceptibility of E. globulus to Mycosphaerella leaf disease it is important to define the leaf pathogen community and the actual species of the pathogen responsible for damage.  This information is required to determine if genetic variation in host tree resistance is pathogen general or pathogen specific.  The main Mycosphaerella species believed to cause severe damage to E. globulus in Tasmania are M. cryptica and M. nubilosa (Table 1).  Mycosphaerella nubilosa infects mainly juvenile foliage whereas M. cryptica may occur on either juvenile or adult foliage. There are also several other species know from E. globulus in Tasmania such as M. parva, M. vespa and M. tasmaniensis (Mohammed et al. 2004).


Table 1. Species of Mycosphaerella leaf disease reported to be causing damage in Australian trials that were used for studying the genetic basis of disease susceptibility in Eucalyptus globulus.

 Study  Trial location
E. globulus host foliage type
Mycosphaerella species1

Dungey et al. 1997

Carnegie and Ades 2002

 NW Tasmania (Ridgley)
juvenile
M. cryptica/
M. nubilosa



adult
M. cryptica
Carnegie and Ades 2002  Victoria adult
M. cryptica
Milgate et al. 2005
 NW Tasmania
(Ridgley)
juvenile
M. nubilosa
Freeman et al. 2008
 NW Tasmania
(Woolnorth)
juvenile
M. cryptica

 SE Tasmania
(Wielangta)
juvenile
M. cryptica/
M. nubilosa
 Present study2
 NW Tasmania
(4 trials)

juvenile
M. cryptica (dominant)/
M. nubilosa


adult
M. cryptica



1 M. cryptica is the dominant cause of damage in both juvenile and adult leaves of E. nitens (Carnegie and Ades 2002).
2 Preliminary assessment based on lesion morphology.

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leaves with cryptic

Lesions on juvenile E. globulus leaves from a single tree that were (top) Mycosphaerella cryptica-like  and (bottom) M. nubilosa-like in their appearance.  Mycosphaerella cryptica, M. nubilosa and M. vespa were detected in the lesions in the top image and M. nubilosa and M. parva detected in the lesions in the bottom image.

It is often difficult to confidently differentiate Mycosphaerella species based on field symptoms such as the morphology of lesions or the distribution fruiting bodies, especially when multiple species are present.  Precise identification can be made by studying the development of germ tubes during ascospore germination, but this is time consuming.  DNA techniques have now been developed which allows the rapid discrimination of the Mycosphaerella species mentioned above (Glen et al. 2007).  This approach is based on what is known as the polymerase chain reaction (PCR) which allows very small quantities of DNA to be amplified, in this case a piece of DNA which reveals variation that is diagnostic of the different Mycosphaerella species.


Dr. Morag Glen (CSIRO) has started DNA testing for the presence/absence of each of the above five Mycosphaerella species in lesion samples taken from random trees in the four northwestern Tasmanian progeny trials of E. globulus in which we are studying the genetics of disease susceptibility.  Morag has also visually classified the lesions which were sampled.  The DNA test has detected all five Mycosphaerella species at all trial sites with the exception of the rarer M. tasmaniensis which is yet to be detected at one site. As expected, M. nubilosa was rarely detected from adult leaf samples whereas M. cryptica generally had a higher incidence on adult foliage. However, based on morphology most of the lesions examined on adult and juvenile leaves were considered to match those of M. cryptica rather than M. nubilosa, suggesting that it is M. cryptica which is causing most of the damage we assessed.


The development of DNA-based techniques to quantify the amounts of each species present ("quantitative PCR") are being developed (Glen et al. 2008), and will be used to check that it is M. cryptica which is responsible for most leaf damage, despite other Mycosphaerella pathogen species occurring even within the same lesion.



References
Carnegie AJ, Ades PK (2002) The proportion of leaf spots caused by Mycosphaerella cryptica and M. nubilosa on Eucalyptus globulus, E. nitens and their F1 hybrids in a family trial in Tasmania, Australia. Australasian Mycologist 21, 53-63.
Carnegie AJ, Ades RK (2005) Variation in Eucalyptus globulus Labill. and E. nitens Dean and Maiden in susceptibility of adult foliage to disease caused by Mycosphaerella cryptica (Cooke) Hansf. Silvae Genetica 54, 174-184.
Dungey HS, Potts BM, Carnegie AJ, Ades PK (1997) Mycosphaerella leaf disease: genetic variation in damage to Eucalyptus nitens, E. globulus and their F1 hybrid. Canadian Journal of Forest Research 27, 750-759.

morag glen with pcr

 Dr Morag Glen sets up PCRs to quantify the relative levels of pathogen specific DNA.

Freeman J, Vaillancourt RE, Potts BM (2008) Few Mendelian genes underlie the quantitative response of a forest tree, Eucalyptus globulus, to a natural fungal epidemic. Genetics 178: 563-571.
Glen M,  Mohammed CL. 2008. Real-time PCR detection ofMycosphaerella spp. infecting eucalypts. Journal of Plant Pathology 90: S2.281
Milgate AW, Potts BM, Joyce K, Mohammed C, Vaillancourt RE (2005) Genetic variation in Eucalyptus globulus for susceptibility to Mycosphaerella nubilosa and its association with tree growth. Australasian Plant Pathology 34, 11-18.
Mohammed CL, Wardlaw TJ, Smith A, Pinkard EA, Battaglia M, Glen M, Tommerup I, Potts BM, Vaillancourt RE (2003). Mycosphaerella leaf diseases of temperate eucalypts around the southern pacific rim. New Zealand Journal of Forest Research. 33, 362-372.