First line of defense: Eucalyptus leaf waxes influence infection by an aggressive fungal leaf pathogen
Abstract
- Leaf epicuticular waxes provide important anatomical and chemical defences against fungi that infect leaves. In this study we analysed the leaf wax composition of Eucalyptus grandis × Eucalyptus urophylla hybrids with contrasting susceptibilities to Teratosphaeria leaf blight (TLB) caused by Teratosphaeria destructans, one of the most important foliar diseases of Eucalyptus.
- The Eucalyptus cuticular wax was extracted from non-inoculated and inoculated genotypes with different levels of susceptibility to TLB and analysed by gas chromatography–mass spectrometry.
- The results showed that a triterpenoid, cycloartenol (CAS), was abundant in a resistant genotype and that hexanedioic acid content increased in the resistant genotypes in response to T. destructans infection. In contrast, palmitic acid was significantly more abundant in the inoculated highly susceptible genotype. In-vitro and in-planta T. destructans spore germination assays with pure compounds, showed that CAS and hexanedioic acid significantly inhibited spore germination. Application of these two compounds to the leaves of a susceptible host also significantly increased resistance to infection. In contrast, palmitic acid promoted spore germination and, when applied to the leaves of a resistant genotype, increased colonization by the pathogen.
- This is the first study providing insights into differences in the leaf wax composition of hosts with different levels of susceptibility to T. destructans. It also showed that leaf wax compounds can modulate spore germination and, ultimately, host resistance to infection.
INTRODUCTION
Leaves, young shoots and fruits of higher plants possess a hydrophobic cuticle layer, formed by the deposition of cutin and occasionally wax on the outer epidermal cell walls (Juniper & Jeffree 1983). Cuticular waxes are chemically diverse, and contain unique mixtures of primary and secondary alkanes, alcohols, aldehydes, ketones, triterpenes and esters derived from very-long fatty acid chains (C20–C34) (Raffaele et al. 2009; Malinovsky et al. 2014). Physically, wax layers are embedded within the cuticle (intracuticular wax) and are also deposited predominantly as crystalloids on leaf surfaces (Martin & Juniper 1970; Barthlott et al. 1998).
The chemodiversity of cuticular waxes has a key function in the adaptation of terrestrial plants to abiotic and biotic challenges (Martin & Juniper 1970; Ziv et al. 2018). For example, drought-tolerant plants generally have thicker cuticles, enriched with long-chain alkanes (Seufert et al. 2022; Sanjari et al. 2001; Xue et al. 2017). In addition, epicuticular waxes play an important role in the host choice of piercing/sucking insects (Begum et al. 2016; Makunde et al. 2023) as well as chewing herbivores (Eigenbrode & Espelie 1995). The most dramatic effects of cuticular waxes can be seen in plant–fungus interactions. Foliar pathogens use the chemical composition of leaf waxes for host recognition, which triggers fungal germination (Feng et al. 2009; Uppalapati et al. 2012), but pathogen-generated breakdown products from the cuticular layer can also facilitate recognition of the pathogen by the host (Fauth et al. 1998). In addition, toxic substances embedded in the cuticle, such as glucosinolates and oxygenated fatty acids, can form a chemical defence barrier against fungal penetration (Ahuja et al. 2016; Santos et al. 2019; Dubey et al. 2020).
On Eucalyptus leaves, the wax crystalloids occur as plates, tubes, or a mixture of plates and tubes (Hallam 1964), but their arrangements and distribution differ significantly between species (Hallam & Chambers 1970; Knight et al. 2004). Interestingly, differences in leaf wax morphology on Eucalyptus leaves have been shown to determine the success of infection by foliar pathogens (Hansjakob et al. 2011; Xavier et al. 2015). For example, urediniospores, germ tubes and appressoria of the rust pathogen, Austropuccinia psidii, had lower viability on Eucalyptus grandis leaves with thick cuticular wax layers (Xavier et al. 2015). However, some Eucalyptus foliar pathogens, such as Quambalaria eucalypti and Teratosphaeria destructans, appear to degrade leaf cuticular waxes during the early infection process prior to stomatal penetration (Pegg et al. 2009; SolÃs et al. 2022).
Teratosphaeria destructans (Capnodiales, Teratosphaeriaceae) is an aggressive pathogen that causes leaf and shoot blight disease on Eucalyptus (Wingfield et al. 1996; Andjic et al. 2019). The disease was first reported in 1995 in Northern Sumatra, Indonesia, affecting young E. grandis trees (Wingfield et al. 1996). Since then, the pathogen has spread rapidly in Eucalyptus plantations throughout tropical and subtropical South East Asia (Andjic et al. 2011; Andjic et al. 2019), including Thailand, East Timor, Vietnam, China, Laos, and Malaysia (Old et al. 2003; Burgess et al. 2006; Barber et al. 2012; Havenga et al. 2021). In 2015, T. destructans was also reported from South Africa on E. grandis × E. urophylla (Greyling et al. 2016) and is now established in Eucalyptus nurseries and plantations in subtropical parts of the country. Despite the spread of T. destructans to many locations in recent years, and the substantial economic losses that it causes in Eucalyptus plantations, the molecular and biochemical mechanisms underlying resistance to the pathogen have not been studied.
The aim of this study was to consider the role of leaf-surface wax composition of Eucalyptus genotypes with different levels of resistance or susceptibility to T. destructans. In addition, the effect of wax compounds on pathogen germination and leaf colonization was evaluated, focusing on compounds that were unique in resistant and susceptible hosts.
MATERIALS AND METHODS
Inoculum preparation
The inoculum was prepared from pure cultures of T. destructans isolate CMW5679 from a E. grandis × E. urophylla host (SolÃs et al. 2022), grown on 2% Malt Extract Agar (MEA, 20 g·l−1) for 3 weeks at 25 °C in the dark, after which the conidial suspension was prepared as described in SolÃs et al. (2022). The suspension was obtained by washing the plates with 20 ml sterile distilled water +0.01% Tween 20 (Sigma-Aldrich, Taufkirchen, Germany). The concentration of the spore suspension was adjusted to 1 × 106 using a haemocytometer.
Plant material and inoculation
Three genotypes of a E. grandis × E. urophylla hybrid (GU2, GU3 and GU4) and a single genotype of E. grandis (G1) were used in this study (kindly provided by SAPPI and MONDI S.A, South Africa). Forty-one-year-old ramets of each of these four genotypes were maintained for 3 months in a greenhouse with an average daily temperature from 20 to 25 °C and average night temperature of 20 °C. Eight healthy plants per genotype were selected for inoculation and leaf epicuticular wax analysis. Four plants per genotype were selected as controls.
The inoculum was sprayed onto both leaf surfaces of the top ten apical leaves, with four replicates per genotype, until run-off. Control plants were sprayed with sterile distilled water +0.01% Tween 20 (Sigma-Aldrich). Plants were maintained in a greenhouse, under natural light with temperatures from 20 to 25 °C. After 10 days, six leaves per replicate of inoculated and non-inoculated (control) plants were harvested for cuticular wax analysis. The remaining four leaves per plant were used to evaluate susceptibility after 35 days using a susceptibility index (SI). SI values were calculated following the scale described in SolÃs et al. (2023). The SI considers the number of leaves affected as well as the severity of the disease (spots, blights, or distortion) on individual leaves (Fig. 1). The susceptibility indices for the four genotypes were as follows: G1 SI = 1.5, GU2 SI = 0.6, GU3 SI = 0.4 and GU4 SI = 1.6 (Fig. 1). Following this SI scale, genotype G1 was classified as susceptible (S), GU2 and GU3 as moderately resistant (MR), and GU4 as highly susceptible (HS) (Fig. 1). G1, GU2 and GU4 showed leaf blight symptomatology, in contrast GU3 was the only genotype that showed isolated leaf spots (Fig. 1). No symptoms were seen on leaves of control plants.
Cuticular wax extraction
The six leaves harvested at 10 days after inoculation from each of the four replicates of E. grandis × E. urophylla genotypes and the E. grandis genotype, either inoculated or non-inoculated, were used to extract cuticular wax, as described in Viana et al. (2010) and modified by Bini (2016). Approximately 1 mg wax was obtained by immersing the six leaves of each replicate in 5 ml chloroform (JT Baker, Phillipsburg, NJ, USA amended with 0.4 μl·ml−1 of the internal standard, 2-phenylethanol, and gently agitating the samples for 30 s. Four biological replicates of non-inoculated and inoculated plants were used for the chemical analyses. The total leaf area was calculated by capturing images of the selected leaves using an Epson Perfection V700 scanner with a resolution of 1200 dots per inch (dpi).
Chemical derivatization and GC–MS analysis of cuticular waxes
The chloroform extract for each of the four inoculated and four non-inoculated samples per genotype were dried at 37 °C under constant air flow and resuspended in 100 μl pyridine containing 20 mg·ml−1 methoxamine HCl. The solution was incubated at 30 °C for 90 min, then centrifuged at 1200 rpm for 20 min. An aliquot of 30 μl of the supernatant was transferred into a glass insert in a glass vial (VWR, Germany). Then, 30 μl MS-TFA (Sigma, USA) were added to the supernatant and incubated at 37 °C for 30 min. A 1 μl sample of the supernatant was analysed on an Agilent 7890 gas chromatograph-mass spectrometer (Agilent, USA) (GC–MS) using a HP5 column with a linear temperature program starting at 70 °C, increasing at a rate of 5 °C·min−1 until a maximum temperature of 300 °C was reached, and then maintained for 2 min. The parameters of the GC–MS: were a solvent delay of 6 min, split inlet with a split ratio of 10:1 and a flow rate of 12 mg·ml−1 leading to a 1.2 ml·min−1 flow rate on the column. The mass spectrometer was set to scan mode, with a low mass of 40 m·z−1 and a high mass of 650 m·z−1 and the ion source was maintained at 70 eV.
Chromatograms were analysed using Agilent MassHunter® Qualitative Analysis software, build 8.0.598.0. Compounds were tentatively identified utilizing the 2017 NIST library (Information Services Office). Data exploration and multivariate principal components analysis (PCA) were conducted using MetaboAnalyst 4.0.
Effect of CAS, hexanedioic acid and palmitic acid on germination in-vitro and in-planta
A bioassay was performed using commercially available cycloartenol (CAS), palmitic acid (hexadecanoic acid) and hexanedioic acid (all Sigma Aldrich). Serial dilutions of derivatized pure standards were analysed to confirm the identity and concentration of the compounds of interest. The starting concentrations were 5.0 ppm, diluted down to 2.0, 1.0 and 0.1 ppm. Compound quantities in leaf wax mixtures were calculated as mg·cm−2 leaf surface area.
The effect of each compound on pathogen germination was evaluated at four different concentrations; 0 (control), 0.1, 1.0 and 5.0 ppm. For the in-vitro assay, each compound was diluted in ethanol (JT Baker) at different concentrations. An aliquot of 1 ml of each solution was evenly distributed on the surface of a 50-mm diameter Petri dish and allowed to air dry in a laminar flow cabinet until the surface was free of visible moisture. A T. destructans conidial suspension (150 μl), prepared as described above, was homogenously distributed onto the base surface of Petri dishes amended with the compounds of interest at each concentration, with four replicates. Because of their hydrophobic nature, the compounds remained fixed to the hydrophobic plastic surface in the presence of an aqueous spore suspension. The Petri dishes were placed into plastic boxes on a rack, suspended 5 cm above sterile distilled water,and sealed for 72 h at 25 °C to maintain a high humidity, after which spore germination was assessed. The germinated conidia were identified when the germ tubes were clearly visible under 20× magnification using an Axioskop 2 plus microscope (Zeiss, Oberkochen, Germany).
For the in-planta assay, each compound was diluted in sterile distilled water to 0 (control), 0.1, 1.0 and 5.0 ppm from an ethanol stock solution of 100 ppm. Approximately 5 ml of each CAS and hexanedioic acid dilution were applied to the adaxial and abaxial surfaces of the eight youngest leaves of four replicate plants of the highly susceptible variety GU4. Similarly, dilutions of palmitic acid were applied to the young leaves of the moderately resistant host, GU3. Subsequently, the leaves were allowed to dry for 2 h. Then, a T. destructans spore suspension of 1 × 106 spore·ml−1 was prepared as described above and applied to the leaf surfaces; the spore suspension was sprayed until run-off onto both the adaxial and abaxial surfaces of the leaves of each experimental plant. Four replicates per treatment were treated as controls, where the eight youngest leaves were sprayed with sterile distilled water. Plants were maintained in a greenhouse, under natural light at temperatures ranging from 20 to 25 °C. Four leaves per individual were harvested at 72 h to determine percentage of pathogen germination and to examine changes in the abundance of the leaf epicuticular wax using scanning electron microscopy (SEM).
For SEM, four leaves per plant were cut into equal 1 cm2 squares and placed in 2.5% glutaraldehyde/formaldehyde for 24 h, as described by SolÃs et al. (2022). Samples were dehydrated using an ethanol series from 30% to 100%, and later placed in hexamethyldisilazane (HMDS) and mounted on aluminium stubs. The samples were then coated with carbon using a Quorum Q150T Coating Unit (Quorum Emitech, London, UK). Conidial germination percentage was estimated in an area of 1 cm2 under a Zeiss 540 Gemini Ultra Plus FEG SEM (Zeiss, 167) at the Laboratory for Microscopy and Microanalysis, University of Pretoria, Pretoria, South Africa. The remaining four leaves in the treatment and controls were maintained for 30 days in the greenhouses under natural light, with a temperature ranging from 20 to 25 °C to determine the new SI values.
Statistical analyses
A completely randomized design was used for all the experiments. Statistical analyses were performed using MetaboAnalyst V.4 and R version 3.2.0 (R Foundation for Statistical Computing, Vienna, Austria). Data were normalized using square-root transformations. The data were analysed statistically for each assay using anova, and Tukey’s post-hoc test was used to determine the significance of differences between all treatments at a 5% confidence level (P < 0.05).
RESULTS
Cuticle wax characterization
The GC–MS analysis revealed more than 300 compounds from the leaf surfaces of the four studied Eucalyptus genotypes. The normalized peak areas were analysed using PCA. The PCA for the ten major compounds showed that the sum of the first two principal components explained 49% of the total variance (Fig. 2A). The inoculated genotypes, GU2, G1, and GU4, and non-inoculated plants clustered together (Fig. 2A). In contrast, the inoculated moderately resistant genotype GU3 formed a separate cluster (Fig. 2A). The main compounds included alkenes (tetradecane, cetene, docosene), esters (benzenepropanoic acid), aromatic hydrocarbons (benzanthracene derivatives), phenols (2,4-di-tert-butylphenol) and fatty acids (octadecanoic; hexadecanoic acid) (Fig. 2B).
Effect of CAS, hexanedioic acid and palmitic acid on germination in-vitro and in-planta
Hexanedioic acid and the triterpenoid CAS were selected for further study based on their higher accumulation in resistant genotypes under pathogen challenge (Fig. 2C, D). Palmitic acid was selected because of its increased concentration in the inoculated susceptible genotypes and its lower concentration in inoculated resistant genotypes (Fig. 2E).
The spore germination of T. destructans, assessed in-vitro under different concentrations of palmitic acid, was significantly higher when applied as a solution of 0.01 ppm (35.75%), 1.00 ppm (44.7%) and 5.00 ppm (42.7%), compared to the control with 0 ppm (21.7%) (Fig. 3A). In-planta, the spore germination in the control was 35.5%. At higher concentrations of palmitic acid, spore germination increased significantly to 50% at 0.01 and 1.00 ppm (P < 0.05) (Figs 3B and 4B). Palmitic acid also increased the susceptibility of the host. When applied to a resistant host (GU3) with an SI of 0.4 (moderately resistant) at 0 ppm the SI increased to 0.6 at 5 ppm, thus changing its status to moderately susceptible (Fig. 5A, D). These results show that palmitic acid promotes T. destructans germination and increases the success of its establishment.
In-vitro treatment with hexanedioic acid resulted in 55.7% spore germination in the control (0 ppm), and this decreased with increased concentrations of the compound to 43% at 0.01 ppm, and was significantly lower at 1 ppm (31%) and 5 ppm (16%) (Fig. 3E). The effect of hexanedioic acid on spore germination in-planta was consistent with results from the in-vitro assay. In this case, the germination at 0 ppm of the compound was 58% and was significantly lower at 0.01 ppm (42%), 1 ppm (39%) and at 5 ppm (20%) (Fig. 3F). Treatment with hexanedioic acid lowered the SI values from 1.6 (0 ppm) to 0.9, 0.85 and 0.4 at 0.1, 1 ppm and 5 ppm, respectively, thereby reducing susceptibility from highly susceptible (1.6), to moderately resistant (SI = 0.4) (Fig. 5C, E). Thus, the in-vitro and in-planta assays both showed that hexanedioic acid reduced T. destructans spore germination and expression of disease symptoms.
Effect of the compounds on epicuticular wax abundance
Scanning electron microscopy of leaves showed that the epicuticular wax on E. grandis × E. urophylla leaves is predominantly deposited in the form of platelets. However, the highly susceptible GU4 genotype had a more glabrous surface morphology (Fig. 4). An incremental increase in platelets was observed in this genotype with increasing CAS concentrations (Fig. 4D). At higher concentrations of CAS, spores in contact with the wax surface remained inert (Fig. 4C, D). In contrast, in the resistant GU3 genotype, the leaves treated with palmitic acid showed a reduction in platelet abundance at the highest concentration of the compound (Fig. 4H). Here, mycelial growth of the pathogen was evident on the leaf surface and stomatal penetration was observed (Fig. 4F–H).
DISCUSSION
This study showed that the composition of the leaf wax surface of E. grandis × E. urophylla genotypes and an E. grandis genotype influence their susceptibility to infection by the aggressive leaf blight pathogen T. destructans. We identified three major chemical compounds on the leaf surfaces, including CAS, hexanedioic acid and palmitic acid, which modulated the spore germination of T. destructans both in-vitro and in-planta. Additionally, our SEM results showed that CAS increased the abundance of leaf wax platelets in a susceptible host. In contrast, palmitic acid reduced platelet abundance. We also demonstrated that these compounds altered host susceptibility, especially in the case of leaves treated with CAS, where a susceptible genotype treated with this compound became resistant to T. destructans infection after treatment.
Evaluation of leaf epicuticular wax composition showed that in all four tested Eucalyptus genotypes, the major leaf wax compounds were fatty acids, such as octadecanoic acid, as well as straight-chain alkenes, such as docosene. This is similar to previous findings in Eucalyptus, where the major components of leaf surface waxes were identified as fatty acids and straight-chain alkenes (Santos et al. 2019; Makunde et al. 2023). Our results showed significant variation between the wax metabolite profiles of different hybrid genotypes. This illustrates the metabolic plasticity of wax synthesis in Eucalyptus and the different putative roles that waxes might have in the recognition of the host by T. destructans and its attachment to the hydrophobic surface of the leaves. Similar effects have also been found for the biotrophic pathogens Blumeria graminis on barley (Zabka et al. 2008) and Magnaporthe grisea on rice (Lee et al. 1994).
Palmitic acid was a highly abundant compound in the inoculated susceptible genotypes included in this study, and its concentration was lower in inoculated leaves of the more resistant genotype. We demonstrated that this compound induced the spore germination of T. destructans in-vitro, as well as on leaves of a resistant genotype in-planta. This fatty acid has been reported previously to induce germination and appressorium differentiation in the rice pathogen Magnaporthe grisea (Gilbert et al. 1996). It is also known to promote conidial germination and cutinase expression of Botrytis cinerea (Leroch et al. 2013). Particularly relevant to this study, it has recently been reported as the major component on the leaf wax surface of a susceptible E. grandis genotype, where it induced germination of urediniospores of the myrtle rust pathogen Austropuccinia psidii (Santos et al. 2019). The mechanism by which this compound enhances host susceptibility is unknown, but it might play a role in host recognition, as has been shown for Blumeria graminis f.sp. tritici (Kong et al. 2020) As a fatty acid, it could also be utilized by fungi as a nutrient source during germination on leaf surfaces, as has been reported for Aspergillus nidulans (Dashti et al. 2008).
The triterpenoid CAS was identified on E. grandis × E. urophylla leaves by GC–MS to be present only in the moderately resistant genotype GU3, and was also more abundant after T. destructans infection. This compound inhibited germination of the pathogen both in-vitro and in-planta. The antimicrobial activity of CAS has been studied in stem bark extracts of the tree Garcinia lucida, and tested against various bacteria and Candida albicans (Momo et al. 2011). CAS is also a major compound of leaves of Garcinia mangostana, and leaf extracts from this plant have antimicrobial activity against the bacterial plant pathogens, Pseudomonas syringae pv. tomato and Xanthomonas oryzae pv. oryzae (Alsultan et al. 2016). The antifungal activity of CAS could relate to the capacity of triterpenoids to interact with the fungal membrane, leading to increased membrane permeability and favouring the entrance of extracellular substances and the leakage of cell constituents, as reported in Candida albicans cells (Haraguchi et al. 1999).
Hexanedioic was found in higher amounts in the wax layer of moderately resistant E. grandis × E. urophylla genotypes after pathogen inoculation. This adipic acid was also shown to reduce T. destructans germination in-vitro and in-planta in a susceptible host. Hexanedioic has also been extracted from leaves of Ficus sycomorus and was linked with insecticidal and acaricidal activity (Romeh 2013). The antifungal activity of hexanedioic acid has been reported from leaf extracts of Melia azedarach against the soil-borne fungal pathogen, Sclerotium rolfsii (Sana et al. 2016). This adipic acid has previously been identified as one of the main compounds in wood extracts of Eucalyptus globulus (Freire et al. 2002). Although the mechanisms of antifungal action are still unknown, it has recently been reported that adipic acids can alter the plasma membrane of Saccharomyces cerevisiae (Fletcher et al. 2021), and this may be related to the mode of action of this compound that inhibited the germination of T. destructans in our study.
The results of this study demonstrate the chemodiversity of the epicuticular wax surface of Eucalyptus leaves, and that it influences susceptibility to infection by T. destructans, as well as spore germination of the pathogen. The results could be used to develop rapid screening methods, such as biomarkers for predicting Eucalyptus susceptibility or resistance to TLB disease. Future work should expand on studying leaf wax composition in different Eucalyptus species, including a larger number of replicates and genotypes. This would lead to a better understanding of the role they could play as preformed and induced defence barriers against pathogens. In addition, studies on the genetic and molecular mechanisms involved in wax metabolite biosynthesis and mode of action of these compounds will contribute to the development of effective molecular tools to breed and select disease-resistant Eucalyptus genotypes.
AUTHOR CONTRIBUTIONS
MS, SN, MJW, and AH conceived the study. MS selected the plant material and pathogen, did the inoculation trials, inhibition experiments, and microscopy. JCJ and MS performed the leaf wax extraction and analysed the samples with GC-MS. AH and MS performed the analysis of GC-MS results and identified the compounds. MS and AH designed the inhibition trials. MS wrote the first draft of the manuscript, which was edited by SN, MJW, and AH. All the authors read and approved the final manuscript.
ACKNOWLEDGEMENTS
We thank members of the Microscopy Centre, Department of Physics at the University of Pretoria, South Africa, particularly Mrs. Erna van Wilpe and Ms. Charity Maepa; Mondi Group. Members of the Tree Protection Co-operative Programme (TPCP) including Sappi and Mondi who provided important plant material; Forestry South Africa, the National Research Foundation (grant number 0318590365) and the DST/NRF Centre of Excellence in Plant Health Biotechnology (CPHB), South Africa are recognized for their financial support. The Chilean Doctoral Fellowship Programme of the National Agency for Research and Development (ANID)/Scholarship Program/DOCTORADO BECAS CHILE/2019-72200511, is thanked for a scholarship provided to the first author.
Source: Plant Biology
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