Identification of mechanisms defining resistance and susceptibility of Camellia plants to necrotrophic petal blight disease : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Manawatū, New Zealand
Species in the genus Camellia, which includes the tea crops, oil-producers and
valuable ornamental plants, have economic and cultural significance for many countries.
The fungus Ciborinia camelliae causes petal blight disease of Camellia plants, which has a
short initial asymptomatic phase and results in rapid necrosis and fall of blooms. Ciborinia
camelliae is a necrotrophic pathogen of the family Sclerotiniaceae, which also includes two
broad-host range necrotrophic pathogens, Botrytis cinerea and Sclerotinia sclerotiorum.
Previously it was shown that some Camellia plants, such as Camellia lutchuensis, are naturally
resistant to petal blight. In order to find molecular mechanisms underpinning this
resistance, a genome-wide analysis of gene expression in C. lutchuensis petals was conducted.
The analysis revealed a fast modulation of host transcriptional activity in response to C.
camelliae ascospores. Interaction network analysis of fungus-responsive genes showed that
petal blight resistance includes increased expression of important plant defence pathways,
such as WRKY33-MPK3, phenylpropanoid and jasmonate biosynthesis. A much-delayed
activation of the same pathways was observed in the susceptible Camellia cultivar, Camellia
‘Nicky Crisp’ (Camellia japonica x Camellia pitardii var. pitardii), suggesting that failure to
activate early defence enables C. camelliae to invade and cause tissue necrosis. Early artificial
induction of defence pathways using methyl jasmonate reduced the rate of petal blight in
susceptible ‘Nicky Crisp’ plants, further verifying the role of a rapid defence activation in
petal-blight resistance. Overall, transcriptomic and functional analysis of the Camellia spp.-
C. camelliae interaction demonstrated that the same plant defence pathways contribute to
both resistance and susceptibility against this necrotrophic pathogen, depending on the
timing of their activation.
To further understand the molecular mechanisms of petal blight resistance, the role
of the phenylpropanoid pathway, identified as a key feature in the transcriptome study
above, was investigated in more detail. This pathway produces various metabolites,
including phenolic acids, aldehydes, and alcohols, which have numerous physiological
functions and also participate in the production of flavonoids and lignin. Resistant C.
lutchuensis was shown to rapidly activate the expression of core phenylpropanoid genes after
treatment with C. camelliae ascospores. LC-MS-based quantification of phenylpropanoid
compounds demonstrated that within the first 6 h of the infection, resistant plants had
already accumulated coumaric, ferulic and sinapic acids, while at 24 hpi, concentrations of
coumaraldehyde, sinapaldehyde, and caffeyalcohol were significantly increased. Thus, I
further hypothesized that the compounds produced by the phenylpropanoid pathway may
have fungistatic activity. Indeed, all tested phenylpropanoids inhibited the growth of C.
camelliae in agar plates with different efficacy. Moreover, the application of
phenylpropanoid compounds, including ferulic and coumaric acids, fully prevented the
formation of petal blight lesions on susceptible Camellia ‘Nicky Crisp’ petals. Taken
together, it can be concluded that the phenylpropanoid pathway may contribute to the early
defence against the petal blight via the rapid production of fungistatic compounds.
Ultimately, these compounds could be used to develop natural antifungal sprays to protect
susceptible Camellia flowers.
The analysis of the C. camelliae secretome using LC-MS/MS detection of proteins
showed that the pathogen produces a large number of carbohydrate-active enzymes in
liquid culture and plant petals. Injection of these proteins induced necrosis not only in
susceptible Camellia petals but also in petals of the resistant species and leaves of non-host
Nicotiana benthamiana. It was proposed that these enzymes can contribute to the virulence of
the pathogen by inducing cell death and facilitating necrosis propagation. Thus, the early
defence responses of resistant Camellia plants may possibly stop the development of C. camelliae before it starts releasing carbohydrate-active enzymes during the necrotrophic step
of the infection.
Overall, the results of this research further expand our understanding of plant-
necrotroph interactions, suggesting that the timing of plant immune responses may be a
crucial factor defining the outcome of the necrotrophic infection.