Role of Extracellular Hydrolytic and Ligninolytic Enzymes as Virulence Factors of the Dematophora necatrix in the Activation of Basal Resistance of Walnut
الموضوعات :
Nima Khaledi
1
,
Mahdi Rezaei
2
,
Farshid Hassani
3
1 - Seed and Plant Certification and Registration Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran
2 - Seed and Plant Certification and Registration Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran
3 - Seed and Plant Certification and Registration Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran
الکلمات المفتاحية: Basal resistance, Defence responses, Phenolics, Soil-borne, Virulence,
ملخص المقالة :
Dematophora necatrix is one of the most destructive soil-borne fungal pathogens causing white root rot disease on walnuts in Iran and worldwide. No walnut rootstock genotypes have shown complete resistance to this disease. This study aims to identify and monitor the prevalence of D. necatrix in traditional and commercial walnut orchards as well as to explore extracellular the hydrolytic and ligninolytic enzymes produced by the fungal isolates plus variations in virulence and aggressiveness in vivo. We also examined the role of biochemical mechanisms, including the activities of oxidative enzymes and defensive compounds involved in the basal resistance of walnuts to the necrotrophic D. necatrix. In this study, we investigated the presence of the causal agent of white rot disease in root and soil samples collected from commercial and traditional gardens in 17 regions of Iran. Then, we analysed the relationship between the activity of extracellular hydrolytic and ligninolytic enzymes produced by D. necatrix isolates together with variations in pathogenicity and aggressiveness in vivo. Next, the activity of guaiacol peroxidase (GPX), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL), and level of defensive compounds were studied at various time points after inoculation by different D. necatrix isolates. All fungal isolates were identified as D. necatrix based on their morphological and molecular characteristics. Analysis of the activity of extracellular hydrolytic and ligninolytic enzymes produced by the fungal isolates revealed a higher activity of these enzymes for isolates presenting higher levels of virulence and aggressiveness in walnut seedlings. The current study suggests the critical role of GPX, PPO, and PAL in walnut resistance against D. necatrix, which was associated with lignification and phenolic production. These results provide novel information about the role of metabolism of phenolic compounds in basal resistance of walnut plant after inoculation by D. necatrix, where the level of defence response depends on the level of pathogenicity of isolates.
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ORIGINAL ARTICLE
Role of Extracellular Hydrolytic and Ligninolytic Enzymes as Virulence Factors of the Dematophora necatrix in the Activation of Basal Resistance of Walnut
Nima Khaledi*, Mahdi Rezaei, Farshid Hassani
Seed and Plant Certification and Registration Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran
K E Y W O R D S |
| A B S T R A C T |
Basal resistance; Defence responses; Phenolics; Soil-borne; Virulence |
| Dematophora necatrix is one of the most destructive soil-borne fungal pathogens causing white root rot disease on walnuts in Iran and worldwide. No walnut rootstock genotypes have shown complete resistance to this disease. This study aims to identify and monitor the prevalence of D. necatrix in traditional and commercial walnut orchards as well as to explore extracellular the hydrolytic and ligninolytic enzymes produced by the fungal isolates plus variations in virulence and aggressiveness in vivo. We also examined the role of biochemical mechanisms, including the activities of oxidative enzymes and defensive compounds involved in the basal resistance of walnuts to the necrotrophic D. necatrix. In this study, we investigated the presence of the causal agent of white rot disease in root and soil samples collected from commercial and traditional gardens in 17 regions of Iran. Then, we analysed the relationship between the activity of extracellular hydrolytic and ligninolytic enzymes produced by D. necatrix isolates together with variations in pathogenicity and aggressiveness in vivo. Next, the activity of guaiacol peroxidase (GPX), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL), and level of defensive compounds were studied at various time points after inoculation by different D. necatrix isolates. All fungal isolates were identified as D. necatrix based on their morphological and molecular characteristics. Analysis of the activity of extracellular hydrolytic and ligninolytic enzymes produced by the fungal isolates revealed a higher activity of these enzymes for isolates presenting higher levels of virulence and aggressiveness in walnut seedlings. The current study suggests the critical role of GPX, PPO, and PAL in walnut resistance against D. necatrix, which was associated with lignification and phenolic production. These results provide novel information about the role of metabolism of phenolic compounds in basal resistance of walnut plant after inoculation by D. necatrix, where the level of defence response depends on the level of pathogenicity of isolates. |
Introduction
*Corresponding author: Email address: n_khaledi@areeo.ac.ir Received: 10 May 2024; Received in revised form: 7 August 2024; Accepted: 10 November 2024 DOI: 10.60680/jon.2024.1119480
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Plants activate different mechanisms to adapt to biotic stresses by changing signal transmission pathways (Hosseini-Zahani and Taheri, 2023). In response to oxidative stress, plants produce enzymatic antioxidants such as guaiacol peroxidase (GPX), as well as non-enzymatic antioxidants such as flavonoids and phenolics to scavenge for different types of reactive oxygen species (ROS) and protect the host (Kuvalekar et al., 2011). The enzyme polyphenol oxidase (PPO) can act as an antioxidant by oxidizing phenolic compounds to quinones (Araji et al., 2014). It can also act as a pro-oxidant by promoting ROS production (Boeckx et al., 2015).
Phenolic compounds and flavonoids are secondary metabolites with multiple functions, such as antioxidant, antifungal, and antimicrobial properties, which are involved in many vital processes of plant growth and development by inducing resistance against different stresses (Khodadadi et al., 2016; Behdad et al., 2020). Most of the antioxidant capacity in walnuts results from phenolic compounds (Arranz et al., 2008; Jariteh et al., 2011; Sarikhani et al., 2021; Amin Ghezel et al., 2022). The level and type of plant secondary metabolites change due to pathogen infection with variations in the activities of defense enzymes (Kuvalekar et al., 2011). The GPX acts as a potent scavenger of H2O2 and participates in oxidizing phenolic compounds, including lignin-derived phenolics (Nowogórska and Patykowski, 2015). Phenylalanine ammonia-lyase (PAL) plays a crucial role as an essential enzyme in the biosynthesis of lignin and phenolic compounds through the phenylpropanoid metabolism pathway (Rasool et al., 2021). Increased PAL and PPO activity enhances the biosynthesis of secondary metabolites in plant tissues, thereby assisting plants in inhibiting pathogen infection (Solekha et al., 2020; Kuvalekar et al., 2011).
Fungal pathogens, belonging to the genus Dematophora, do not have specialized structures to penetrate plant cells (Kubicek et al., 2014) and directly penetrate the epidermal cell walls with short infection hyphae (Pliego et al., 2009). D. necatrix produces various extracellular hydrolytic enzymes, such as cellulase, xylanase, and pectinase, during the infection process of avocado roots (Kim et al., 2017). The root rot pathogen of D. necatrix wields a versatile arsenal of enzymes via which it directly attacks the lignin barrier (Di Lella et al., 2022). White root rot fungi produce the most abundant ligninolytic enzymes, including laccase, manganese, and lignin peroxidase (Urbanek et al., 2018). The cell wall degrading enzymes are unable to degrade lignin on their own and help the lignin degradation by providing H2O2 for the ligninolytic enzymes and reduce the generated intermediates (Chan et al., 2020). Increased activity of extracellular enzymes secreted by plant pathogens is directly linked to increased virulence and invasion of the host plant (Gawade et al., 2017).
To our knowledge, there is no information on the role of defence mechanisms in the basal resistance of walnut to D. necatrix. Further, we have yet to investigate the comparative role of extracellular hydrolytic and ligninolytic enzymes produced by D. necatrix isolates and their association with pathogenicity plus aggressiveness. Thus, the objectives of this research were to: (i) determine the health status of commercial and traditional orchards to white root rot disease, (ii) evaluate the correlation between various extracellular hydrolytic and ligninolytic enzymes as virulence factors of the D. necatrix and their role in the appearance plus progression of disease symptoms in walnut plants, and (iii) investigate changes in defence-related enzyme activities (GPX, PPO, and PAL) as well as levels of defensive compounds (total phenolic, flavonoid and lignin contents) as a part of defence biochemical mechanisms involved in this pathosystem.
Materials and Methods
Sample screening and collection
During the 2023-24 growing season, a total of 71 walnut plants with and without foliage yellowing and wilting symptoms throughout the entire plant or in just a portion of the canopy and fine white mycelia layer on collar and/or root were sampled from commercial and traditional orchards in different regions of Iran (Table 1). For all plants from which root samples were collected, a soil sample (about 1 kg) was also taken by mixing different sub-samples of soil from the rhizosphere at a depth of 5-30 cm. Then, the samples were individually placed in paper bags and transported in an ice box to the laboratory at the Seed and Plant Certification and Registration Institute (SPCRI) of Iran to isolate putative pathogens.
Fungal isolation and morphological identification
For isolation of fungal isolates from the collected root samples, we cut small sections of root tissue (approximately 1 × 1 cm), surface-sterilized in 2% sodium hypochlorite solution for 15 minutes, washed in sterile distilled water and left to air dry (Castro et al., 2013). These small sections were placed in Petri plates containing potato dextrose agar (PDA) amended with lactic acid (0.2%, v/v). Isolates from soil were obtained by the serial dilution plating method (Waksman, 1922). One gram of each soil sample was diluted in 9 mL of sterile distilled water. Then, 1 mL was taken from dilution and transferred to the second test tube. The dilutions continued until the 4th tube, and finally 0.5 mL from 4th tube was spread in Petri dishes containing PDA modified with lactic acid (0.2%, v/v) (Arjona-López et al., 2019). PDA plates were incubated at 25 ± 1 °C in dark for 10 days. Dematophora spp. colonies were identified initially under the light microscope (Olympus BX51) by the presence of the typical hyphal pear-shaped swellings near the septa (Pliego et al., 2012; Pérez-Jiménez, 2006).
DNA extraction, PCR amplification, and molecular identification
Total genomic DNA was extracted using the Genomic DNA isolation kit (Pishgam Biotech, Iran). Validation of morphological features of fungal isolates at the identified species level was performed using the species-specific primers of D. necatrix (forward: 5’-CAA AAC CCA TGT GAA CAT ACC A-3‘and reverse: 5’-CCG AGG TCA ACC TTT GGT ATA G-3‘) according to Schena et al. (2002). The PCR reaction was performed in a volume of 25 μl, each reaction containing 1 μL of 2.5 μM every reverse and forward primers, 12.5 μL of PCR Master Mix (CinnaGen, Iran), and 3 μL of sample DNA and 7.5 μL PCR grade water. The PCR program involved a pre-incubation step for 5 min at 94 ◦C followed by 40 cycles of 45 s at 94 ◦C, 45 s at 60 ◦C, 60 s at 72 ◦C and final extension for 5 min at 72 ◦C. Amplified products were separated via gel electrophoresis in a 1.5% agarose and visualized with GelRed staining on a UV gel documentation system (Syngene Gene Flash Bio, USA). Negative control with molecular grade water instead of template DNA and positive control containing DNA of D. necatrix in PCR reaction mix were included with all PCR runs.
Plant materials
Persian walnut Damavand (Iranian and local walnut cultivar) was used for virulence tests. ‘Damavand’ is a widespread walnut genotype planted in many walnut-growing regions of Iran because of its promising growth and high yield under Iran's climatic conditions. Walnut seedlings were planted individually in each of the 15 cm diameter (× 11 cm height) plastic pots containing sterilized mixture of peat moss, perlite, and field soil at 1: 1: 1 (v/v/v). The plants were grown under greenhouse conditions at 30 ± 4 °C; with a 16/8h light/dark photoperiod; 50 ± 5 % relative humidity; and irrigated when needed.
Virulence test on walnut plants
D. necatrix inoculum preparation
Fungal inoculum was prepared on autoclaved wheat grains as described by Freeman et al. (1986). In each pot, we carried out artificial inoculation using 30.0 g of colonized grains per approximately 1.0 kg of substrate soil. To ensure the spread of the inoculum, we placed it across four points distributed around the basal stem and root crown (~ 2.5 cm apart) and introduced it at two depths (~5 cm and ~ 10 cm, respectively). The sterilized wheat grains without the fungus served as the control.
Assessment of aggressiveness
To confirm Koch’s postulates, fungal isolates were tested for virulence on walnut plants under controlled conditions. In the experimental design, four replicate walnut trees were planted for each isolate in a completely randomized design and the experiment was repeated twice. Aerial symptoms including chlorosis and defoliation were observed 21 days after inoculation, while control plants remained symptomless. Disease severity was evaluated by measuring the aerial symptoms of white root rot using a 0 (healthy) to 5 (plant died) scale as described Sun et al. (2008). Finally, the disease severity index (DSI) was calculated using the following formula (Khaledi et al., 2021):
DSI% = [∑(Number of diseased plants at all levels) Í (The value of relevant level) / (Total number of investigated plants) Í (Highest disease level)] Í100
Aggressiveness, as a quantitative component of virulence, was studied for each fungal isolate on walnut seedlings using the method described by Kleina et al. (2019) based on determining hours post-inoculation (HPI) for the onset of disease symptoms.
Extraction and enzymatic analyses
Five mycelial plugs (5 mm diameter) from each fungal culture actively growing on a PDA plate were transferred to 250 mL Erlenmeyer flasks containing 100 mL of sterile in primary culture media of hemicellulase (xylanase), pectinase, cellulose, and ligninolytic activities as described by other researchers (Khaledi and Hassani, 2018; Koroljova-Skorobogat’ko et al., 1998). Then, the flasks were incubated at 25 ± 2 °C on a rotating shaker at 120 rpm for 10 days in vitro. In order to obtain the crude enzyme solution, we filtered a volume of 5 mL of the medium in the flask at various times through a Whatman No.1 filter paper. Afterward, the obtained filtrate was centrifuged at 10,000 rpm at 4 °C for 20 min and then used the supernatant to determine enzyme activities. Xylanase activity was determined according to the method described by Bailey et al. (1992). Pectinase activity was determined according to the method described by Kaur and Gupta (2017), based on the amount of reduced D-galacturonic acid released in the culture supernatant. Cellulase activity was determined according to the method described by Wood and Bhat (1988). Ligninolytic enzymes, such as laccase, lignin peroxidase, and manganese peroxidase, were mainly secreted by white rot fungi, which can effectively degrade lignin. Therefore, we studied the activity of laccase, lignin, and manganese peroxidase enzymes. Laccase activity was determined according to the method described by other researchers (Praveen et al., 2011; Das et al., 1997; Kuwahara et al., 1984).
Determination of defence enzyme activities
The plant aerial parts (500 mg) were ground collected from various treatments [seedling uninoculated, seedling inoculated by D. necatrix isolates MU57, RV08, ZA94, LX66, FP35, ND36, BE71, CT17, GD23, SC89 and HO42] into a fine powder with a mortar and pestle under liquid nitrogen, then homogenized in 2 ml of a solution containing 50 mM potassium phosphate buffer (pH 6.8), 1 mM ethylenediaminetetraacetic acid (EDTA), 2% (w/v) polyvinylpyrrolidone (PVP), and 1 mM phenylmethylsulfonyl fluoride (PMSF). The homogenate was then centrifuged at 12,000 g and 4 °C for 15 min to extract the supernatant for enzymatic analysis (Almeida et al., 2024). Sampling of root tissues was performed at various time points following inoculation by different D. necatrix isolates [1, 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 days post-inoculation (DPI)]. The method of Bradford (1976) was used to determine the total protein concentration, with bovine serum albumin (Sigma) serving as a standard.
Guaiacol peroxidase (GPX) activity was assessed according to Konieczny et al. (2014) and was expressed as μmol tetraguaiacol.min-1 mg-1 protein. Polyphenol oxidase (PPO) activity was measured according to Mayer et al. (1965) and was expressed as μmol.min-1 mg-1 protein. Phenylalanine ammonia-lyase (PAL) activity was determined based on the rate of cinnamic acid production as described by Ramamoorthy et al. (2002) and was expressed as nmol trans-cinnamic acid.min-1 mg-1 protein.
Determination of total phenolic, total flavonoid, and lignin contents
The total phenolic content was determined using the Folin-Ciocalteu reagent method as described by Wang et al. (2022). Total flavonoids and lignin content were determined according to the method of Zhang et al. and Suzuki et al. (2009). All experiments were performed in triplicate and repeated twice.
Statistical analysis
The obtained data were statistically analysed via SAS software (version 9.2; SAS Institute, Cary, NC, USA) and mean comparison was conducted using the Duncan’s multiple range test at the level P ≤ 0.05.
Results
Morphological and molecular identification of Dematophora isolates
A total of 142 samples including 71 soil samples and 71 root samples from approximately 17 walnut orchards in various regions of Iran (Table 1) were collected for screening the causal agent of white root rot disease (on average 6 to 14 samples per orchard plot). In this study, 11 fungal isolates were recovered from root and soil samples in the studied orchards. Eight isolates were isolated from root sections of walnut trees collected in four traditional orchards (Gorgan, Asadabad, Sahneh, and Chenaran). Three isolates were isolated from soil samples collected in three traditional orchards (Gorgan, Asadabad, and Sanandaj). Among the samples collected from the soil and roots, no Dematophora infection was observed and detected in all commercial orchards (Kuhin and Tuyserkan), and ten traditional orchards (Mobarakeh, Urmia, Damavand, Bojnord, Shahrud, Marvdasht, Jiroft, Shahzan, Joveyn, and Azarshahr) from different regions of Iran. The results of this research indicated that four, three, two, one, and one fungal isolates recovered of Gorgan, Asadabad, Chenaran, Sanandaj and Sahneh samples, respectively (Table 1). Among the samples collected from traditional orchards in the studied regions, the Gorgan sample showed the highest frequency of Dematophora isolates obtained from the collected soil and root samples (Table 1).
Table 1. Characteristics of soil and root samples based on sampling site and frequency of Dematophora isolates identified in traditional and commercial walnut orchards.
Orchard type | Orchard code | Sample site | NDI* | Orchard type | Orchard code | Sample site | NDI* | |||||||
Soil sample | Root sample | Soil sample | Root sample | |||||||||||
Commercial orchards | KQ67 | Qazvin - Kuhin | ND | ND | Traditional orchards | BK92 | North Khorasan - Bojnord | ND | ND | |||||
Qazvin - Kuhin | ND | ND | North Khorasan - Bojnord | ND | ND | |||||||||
Qazvin - Kuhin | ND | ND | North Khorasan - Bojnord | ND | ND | |||||||||
Qazvin - Kuhin | ND | ND | SS13 | Semnan - Shahrud | ND | ND | ||||||||
Qazvin - Kuhin | ND | ND | Semnan - Shahrud | ND | ND | |||||||||
Qazvin - Kuhin | ND | ND | Semnan - Shahrud | ND | ND | |||||||||
Qazvin - Kuhin | ND | ND | MS56 | Fars - Marvdasht | ND | ND | ||||||||
TH91 | Hamadan - Tuyserkan | ND | ND | Fars - Marvdasht | ND | ND | ||||||||
Hamadan - Tuyserkan | ND | ND | Fars - Marvdasht | ND | ND | |||||||||
Hamadan - Tuyserkan | ND | ND | JK35 | Kerman - Jiroft | ND | ND | ||||||||
Hamadan - Tuyserkan | ND | ND | Kerman - Jiroft | ND | ND | |||||||||
Hamadan - Tuyserkan | ND | ND | Kerman - Jiroft | ND | ND | |||||||||
Hamadan - Tuyserkan | ND | ND | UA05 | West Azerbaijan - Urmia | ND | ND | ||||||||
Hamadan - Tuyserkan | ND | ND | West Azerbaijan - Urmia | ND | ND | |||||||||
Traditional orchards | SM21 | Markazi - Shahzan | ND | ND | West Azerbaijan - Urmia | ND | ND | |||||||
Markazi - Shahzan | ND | ND | JH73 | Razavi Khorasan - Joveyn | ND | ND | ||||||||
Markazi - Shahzan | ND | ND | Razavi Khorasan - Joveyn | ND | ND | |||||||||
SK23 | Kurdistan - Sanandaj | 1 | ND | Razavi Khorasan - Joveyn | ND | ND | ||||||||
Kurdistan - Sanandaj | ND | ND | AG44 | East Azerbaijan - Azarshahr | ND | ND | ||||||||
Kurdistan - Sanandaj | ND | ND | East Azerbaijan - Azarshahr | ND | ND | |||||||||
AH18 | Hamadan - Asadabad | ND | ND | East Azerbaijan - Azarshahr | ND | ND | ||||||||
Hamadan - Asadabad | ND | 2 | CK87 | Razavi Khorasan - Chenaran | ND | ND | ||||||||
Hamadan - Asadabad | ND | ND | Razavi Khorasan - Chenaran | ND | ND | |||||||||
Hamadan - Asadabad | 1 | ND | Razavi Khorasan - Chenaran | ND | ND | |||||||||
Hamadan - Asadabad | ND | ND | Razavi Khorasan - Chenaran | ND | 1 | |||||||||
Hamadan - Asadabad | ND | ND | Razavi Khorasan - Chenaran | ND | ND | |||||||||
Hamadan - Asadabad | ND | ND | Razavi Khorasan - Chenaran | ND | ND | |||||||||
SK04 | Kermanshah - Sahneh | ND | 1 | Razavi Khorasan - Chenaran | ND | 1 | ||||||||
Kermanshah - Sahneh | ND | ND | GG50 | Golestan - Gorgan | ND | 1 | ||||||||
Kermanshah - Sahneh | ND | ND | Golestan - Gorgan | ND | ND | |||||||||
MI96 | Isfahan - Mobarakeh | ND | ND | Golestan - Gorgan | ND | ND | ||||||||
Isfahan - Mobarakeh | ND | ND | Golestan - Gorgan | ND | ND | |||||||||
Isfahan - Mobarakeh | ND | ND | Golestan - Gorgan | ND | 1 | |||||||||
DT62 | Tehran - Damavand | ND | ND | Golestan - Gorgan | 1 | 1 | ||||||||
Tehran - Damavand | ND | ND | Golestan - Gorgan | ND | ND | |||||||||
Tehran - Damavand | ND | ND |
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The morphological characterization revealed that, all fungal isolates were classified only in the Dematophora genus. Fungal isolates form white mycelia with pear-shaped swellings near the septa in the hyphae, which is a diagnostic characteristic of Dematophora necatrix (Pliego et al., 2012; Pérez-Jiménez, 2006). The colony colour of the isolates varied from white to darker grey. The conidia were smooth, one-celled, hyaline, and elliptical shaped, measuring 3.6-4.8 × 1.9-2.2 μm. The conidiophores were branched, erect, brown, 2.2-2.6 μm wide, and more than 400 μm long. After presumptive identification, the fungal isolates were submitted to molecular identification by species-specific PCR. PCR amplification results confirmed that the morphological identification of D. necatrix isolates was performed correctly. All fungal isolates were identified by PCR amplification with D. necatrix-specific primers R2 and R8, which yielded about 493 bp.
Virulence and aggressiveness assay
The results indicated that various D. necatrix isolates on walnut seedlings had various virulence capabilities (Fig. 1a). Based on the mean comparison of disease severity index, all D. necatrix isolates were found to be pathogenic or weakly pathogenic. The Koch’s postulates were demonstrated by the re-isolation of the same fungus from the diseased roots. The results of virulence tests of different D. necatrix isolates on walnut seedlings indicated that the highest and lowest disease index values were recorded for the isolates of MU57 and HO42, respectively (Fig. 1a). The virulence test indicated that the disease index for fungal isolates ranged from 88.5 ± 1.5 to 16.5 ± 2.0 (Fig. 1a).
The data obtained from the aggressiveness test presented that the early development of disease symptoms by isolate MU57 (288 HPI) was comparable to that of the other tested isolates (Fig. 1b). Further, the results indicated that the lowest aggressiveness belongs to the HO42 isolates (420 HPI) (Fig. 1b). The results of the seedling infection tests showed different levels of aggressiveness of the isolates of D. necatrix. According to the results, symptoms of root disease appeared after 12 days post-inoculation (288 HPI) in inoculated plants, which died ultimately 86 days (2,068 HPI) after pathogen inoculation.
Fig. 1. Virulence (a) and aggressiveness (b) of Dematophora necatrix isolates inoculated on seedlings of walnut genotype Damavand.
Analysis of extracellular hydrolytic and ligninolytic
enzyme activities
The study of extracellular enzyme activity revealed that different isolates of D. necatrix had different levels of enzyme activity in vitro (Fig. 2). The enzyme activity levels among the D. necatrix isolates ranged from 355 to 211 µg mL-1 for cellulase, from 511 to 322 µg mL-1 for xylanase, from 782 to 676 µg mL-1 for pectinase, from 77 to 17 µg mL-1 for laccase, from 22 to 9 µg mL-1 for manganese peroxidase, and 146 to 95 µg mL-1 for lignin peroxidase (Fig. 2). The peak time of enzyme activity among D. necatrix isolates varied from 72 to 96 hours post-culturing on liquid medium (HPC) for cellulase, from 120 to 144 HPC for xylanase, from 192 to 216 HPC for pectinase, from 168 to 192 HPC for laccase, from 168 to 216 HPC for manganese peroxidase, and from 144 to 168 HPC for lignin peroxidase in liquid medium (Fig. 2). Isolate MU57 had the highest enzymatic activity in vitro compared to the other tested isolates. The isolate HO42 exhibited the lowest levels of extracellular hydrolytic and ligninolytic enzyme activities (Fig. 2).
In general, the highest level of activity was observed for pectinase, followed by xylanase, cellulase, laccase, lignin, and manganese peroxidase. We compared the activity of extracellular enzymes produced by D. necatrix isolates to examine a potential association between extracellular enzymes and virulence. Isolates HO42 and MU57, which showed the lowest and highest levels of extracellular hydrolytic and ligninolytic enzyme activities, respectively, presented the lowest and highest levels of pathogenicity in walnut seedlings (Fig. 1, Fig. 2).
Fig. 2. Analysis of extracellular hydrolytic and ligninolytic enzyme activity produced by Dematophora necatrix isolates during a 240-hour incubation period. Cellulase activity (a), xylanase activity (b), pectinase activity (c), laccase activity (d), manganese peroxidase activity (e), lignin peroxidase activity (f). The bars indicate standard errors (SE), Values are means of three replicates. 
MU57; 
RV08; 
ZA94; 
LX66; 
FP35; 
ND36; 
BE71; 
CT17; 
GD23; 
SC89; 
HO42
Determination of antioxidant enzymes and phenylalanine
ammonia-lyase activities
We examined the antioxidant activity of GPX and PPO to determine the time point of induction and their role in the basal resistance of walnuts against D. necatrix (Fig. 3). To study the biochemical defence mechanisms involved in basal resistance, we inspected PAL activity as key markers of the phenylpropanoid pathway. A comparative analysis of GPX, PPO, and PAL activities revealed that all plants inoculated with D. necatrix contained significantly higher activity than the uninoculated control at various time points. The highest activities of GPX, PPO, and PAL were detected for walnut plants inoculated with the HO42 isolate at most times studied (Fig. 3). The lowest activities of GPX, PPO, and PAL were observed for plants inoculated with
Fig. 3. Analysis of Guaiacol peroxidase (GPX; a), polyphenol oxidase (PPO; b) and phenylalanine ammonia lyase (PAL; c) activities in walnut genotype seedlings at different times after inoculation with Dematophora necatrix isolates. Data are means ± standard error. The experiment was repeated twice with similar results. Abbreviations in figure: control 
seedling uninoculated, seedling inoculated by D. necatrix isolates MU57; RV08; ZA94; LX66; FP35; ND36; BE71; CT17; GD23; SC89; HO42
MU57, presenting the highest virulence among the D. necatrix isolates used in this research (Fig. 3). GPX and PAL activity increased up to 6 DPI. They dropped up to 12 DPI, followed by an ascending trend in enzyme activity up to 15 DPI and finally, diminished up to 30 DPI (Fig. 3a, Fig. 3c). Plants with different treatments showed peak GPX and PAL activities at 15 DPI. However, the PPO activity continued to grow until nine DPI. It decreased until 15 DPI, followed by an upward trend of enzyme activity until 18 DPI, and finally, it fell until 30 DPI (Fig. 3b). However, in control plants without fungal inoculation, no significant changes were observed in GPX, PPO, and PAL activities and the plant showed a steady state during the period examined (Fig. 3).
Investigating the accumulation of defensive compounds
Our data indicated a significant difference in total phenolic, flavonoid, and lignin content between the different treatments used in this study (Fig. 4). The accumulation of flavonoid and phenolic compounds significantly increased after inoculating the walnut plants with D. necatrix, as compared to the non-inoculated controls. Significant differences were observed among the walnuts treated with different isolates of D. necatrix regarding the relative increase and the timing of maximum accumulation of defensive compounds. The accumulation of defensive compounds increased in response to the walnut-D. necatrix interaction.
The results demonstrated a significantly higher accumulation of total phenolic, flavonoid, and lignin in walnuts treated with isolate HO42 compared to other treatments.
The results revealed that the plant treated with isolate HO42 exhibited increased phenolic and flavonoid contents until six DPI, followed by a decline until nine DPI. Subsequently, there was a rising trend from 9 to 15 DPI, ultimately decreasing to 30 DPI (Fig. 4a, Fig. 4b). The highest level of lignification belonged to the plant treated with isolate HO42 at 21 DPI (Fig. 4c). The accumulation levels of total phenolic, flavonoid, and lignin contents among plants treated with the different D. necatrix isolates varied from 563 to 495 mg GAE. g-1 DW for phenolic content, 283 to 379 mg QE. g-1 DW for flavonoid content, and 62 to 35 mg alkali lignin. g-1 DW for lignin content (Fig. 4).
The least accumulation of phenolic compounds was observed for plants inoculated with the MU57 isolate, presenting the highest pathogenicity among the D. necatrix isolates used in this study. For the walnut treated with the MU57 isolate, the peak of phenolic and flavonoid content was observed at 15 DPI, followed by a descending trend for the rest of the time points tested (Fig. 4a, Fig. 4b). The peak of lignin content was observed at 18 DPI and gradually fell thereafter (Fig. 4c). Plants without fungal inoculation exhibited a consistent level of defensive compounds at the lowest level throughout the study (Fig. 4).
Fig. 4. Levels of total phenolic (a), flavonoid (b) and lignin (c) content in seedling of walnut genotype plants at different times after inoculation with Dematophora necatrix isolates. Data are means ± standard error. The experiment was repeated twice with similar results. Abbreviations in figure: control seedling uninoculated, seedling inoculated by D. necatrix isolates MU57; RV08; ZA94; LX66; FP35; ND36; BE71; CT17; GD23; SC89; HO42
Discussion
This study is the first report on the biochemical mechanisms involved in the basal resistance of walnuts to the necrotrophic fungus D. necatrix. The role of plant defence responses, including the accumulation of phenolic compounds and flavonoids, lignification and induction of GPX, PPO and PAL activities in the pathosystem of walnut and D. necatrix, has been demonstrated in this research. In addition, this is the first detailed study on the relationship between virulence, aggressiveness, and the activity of extracellular enzymes produced by D. necatrix which have been evaluated and compared.
In this study, 11 fungal isolates from different traditional orchards in Iran were isolated and identified using molecular and morphological characteristics. Similar findings were also reported by Behdad (1975), who demonstrated that the fungal isolates were isolated from the diseased roots of walnut plants in the Isfahan orchards of Iran and were identified as D. necatrix (previously Rosellinia necatrix Berl. ex Prill.) based on morphological characteristics. Our observations revealed that pyriform hyphal swellings form at the junctions of septa in mature hyphae (older than seven days) and not in young mycelia, as reported by others (Pliego et al., 2012; Pérez-Jiménez, 2006). Similar results of the morphological and molecular identification of D. necatrix were obtained in nurseries and orchards of different fruit crops by Fusco et al. (2022), Arjona-López et al. (2019), Pliego et al. (2012), Pliego et al. (2009), Takemoto et al. (2011), and Ruano-Rosa et al. (2007). The genus Rosellinia has recently been segregated from Dematophora based on multi-locus phylogenetic analysis, morphological characteristics and considering the “One Fungus - One Species” concept (Suwannasai et al., 2023; Wittstein et al., 2020). Thus, according to the new research, the revival of Dematophora is established for the taxon D. buxi (Pourmoghaddam et al., 2023), D. necatrix (Fusco et al., 2022; Pourmoghaddam et al., 2022), D. pepo (Wittstein et al., 2020) and D. bunodes (Wittstein et al., 2020).
The Dematophora infection was only observed from samples collected in the traditional orchards. Our results showed that the highest frequency of fungal isolates was observed in the Gorgan region, followed by the Asadabad, Chenaran, Sanandaj, and Sahneh regions. This incidence of Dematophora may be attributed to cultivated sensitive genotypes, poor management of irrigation water, improper drainage, environmental conditions, lack of proper orchard management practices, presence of fungal infection in soil, and not using the proper fungicide causing the high incidences of fungal in the traditional orchard of Gorgan region. The rate of spread of D. necatrix varies across studied walnut orchards. Some orchards remain uninfected, while others experience rapid development of diseased and dying plants. Still other orchards have only one or two affected plants (Carlucci et al., 2013; Pérez-Jiménez, 2006), which is in line with our observation in this research.
Pathogen aggressiveness, an elementary quantitative trait, establishes the pathogen’s potential for causing pathogenicity and yield losses on the host (Khaledi et al., 2021). Furthermore, aggressiveness is essential to understand host-pathogen interactions in the pathosystem of walnut and D. necatrix. Our research has presented different levels of virulence and aggressiveness among different isolates of D. necatrix. This finding aligns with the observations made by other researchers (Fusco et al., 2022; Kleina et al., 2019; Pasini et al., 2016; López et al., 2008).
The complex virulence factors of D. necatrix are not well understood, though researchers suggest that the production of extracellular hydrolytic enzymes and secondary metabolites contributes to the infection process (Shimizu et al., 2018; Pliego et al., 2012). Ghatge et al. (2020) reported that the fungus D. necatrix can degrade lignin components. We focused on a detailed investigation of xylanase, cellulase, pectinase, laccase, manganese peroxidase, and lignin peroxidase activities at different time points as these are the main hydrolytic enzymes produced by D. necatrix (Pointing et al., 2003). During the 240-hour study of the extracellular hydrolytic and ligninolytic enzymes, the rate of production and the time required for each enzyme to reach maximum activity differed between the different isolates of D. necatrix. The maximum activity of the extracellular enzymes studied was observed for the isolate MU57, presenting the highest levels of virulence and aggressiveness. Cellulase was the first enzyme to reach the peak, while the maximum activity of the other enzymes was found to be delayed. Schwarze (2007) obtained similar results, observing that Armillaria spp. primarily decays the cellulose, xylanase, and pectin components of the plant cell wall while leaving lignin untouched during the early stages of decay. Other researchers obtained similar results, demonstrating that D. necatrix has a high level of cellulolytic enzyme activity compared to pectinase (Pérez-Jiménez, 2006). The extracellular oxidative enzymes showed maximum activity levels at different periods of incubation time in vitro, which was in line with those of Huang et al. (2017). Furthermore, Martínez et al. (2002) reported that lignin peroxidase activity reaches its maximum level after a short incubation time compared to manganese peroxidase and laccase. This finding aligns with our observation. Similar results were also reported by Sousa et al. (2019). They mentioned that lignin peroxidase was the main lignin-modifying enzyme with a far higher activity than manganese peroxidase and laccase. Song et al. (2022) demonstrated that the degradation of lignin and cellulose does not rely on a single enzyme but rather on the interactions of multiple enzymes, aligning with our findings.
The extracellular enzymes produced by D. necatrix can degrade lignocellulose constituents of plant cell walls (Ruiz-Dueñas et al., 2013). Therefore, it is difficult to determine the role of these secreted extracellular hydrolytic and ligninolytic enzymes in virulence because of functional redundancy. The comparison of extracellular enzyme activities and virulence of the isolates indicates that cellulase and lignin peroxidase had a more significant impact on virulence compared to other hydrolytic and ligninolytic enzymes studied in this research, which is parallel to other reports (Villavicencio et al., 2020; Pérez-Jiménez, 2006). Thus, we found a relationship between the virulence plus aggressiveness of walnut plants and the hydrolytic as well as ligninolytic enzymes produced by D. necatrix isolates in vitro. These results are similar to those obtained by Kim et al. (2017). Our in vitro and in vivo observations have shown that the levels of extracellular enzyme activity correlate with the variation of virulence and aggressiveness of D. necatrix. Therefore, these results suggest that extracellular hydrolytic and ligninolytic enzymes play a crucial role in disease progression. This is the first report demonstrating a strong correlation between the level of extracellular enzyme activity and the virulence of D. necatrix isolated on the host plant.
In order to address the destructive impact of biotic stresses, plants employ various defence mechanisms, such as antioxidant enzymes and defensive compounds (Hosseini-Zahani and Taheri 2023). The current study has revealed that the basal resistance of walnuts against D. necatrix is associated with changes in the levels of defense enzymes (GPX and PPO) and the accumulation of defensive compounds synthesized in the phenylpropanoid pathway. GPX is an essential type of peroxidase, which uses guaiacol as a specific electron donor (Sharma et al., 2012) and occurs in the presence of stressors (Fijalkowski and Kwarciak-Kozlowska, 2020). GPX may play a role in the production of phenolic, serving as an efficient resistance mechanism (Nikraftar et al., 2013). Walnut plants showed the first symptoms of the disease 12 days post-infection. The GPX activity increased significantly at six DPI and 15 DPI, which revealed the role of GPX in diminished oxidative stress and symptoms of white root rot in the walnut. When biotic stress affects the plant, PPO activity redirects phenolics to produce quinone, thereby inhibiting colonization of the tissues by the pathogen (Kakueinezhad et al., 2018). PPO activity increased significantly at 9 and 18 DPI, which highlights the role of PPO in disease resistance of walnut to D. necatrix. Araji et al. (2014) reported that PPO activity in walnut is associated with non-enzymatic ROS scavenging and acts as an indirect antioxidant regulator of cell death, aligning with our observations. Numerous studies have shown that PAL is essential for the resistance of plants to various abiotic and/or biotic stresses (Zhang et al., 2022; Amri et al., 2021). The PAL activity significantly increased at six DPI and 15 DPI, highlighting the vital role of PAL in the plant’s resistance to stress. This result is consistent with the observations of Yan et al. (2019). Tomás-Barberán and Espín (2001) reported that the higher amount of phenols and increase in PAL and PPO activity in plants are attributed to the pathogen infection, which is supported by our results. Our results indicate that the activities of defence enzymes increase in walnut trees after inoculation with D. necatrix. Thus, this increase in defence enzyme activity may be related to the biosynthesis of secondary metabolites in plant defence pathways, which is consistent with the research of Mandal et al. (2010). The phenylpropanoid pathway produces secondary metabolites known as phenolic and flavonoid compounds (Lin et al., 2016). The defence compounds can act through several mechanisms, including scavenging radicals, reducing the effects of oxidative stress, and forming a physical barrier against infection. This protects plants from biotic agents and abiotic stresses (Treutter, 2006). Lignin, a phenolic complex polymer, is a primary defence strategy to hinder the advancement of pathogens in infected tissue (Choi et al., 2023). The data obtained revealed that the total content of phenolics, flavonoids, and lignin increased after infection with D. necatrix isolates compared to uninfected plants. These observations suggest that increased defensive compounds in walnut plants may be a defence strategy against D. necatrix. Similar results were obtained by Jiang et al. (2019), who reported that the phenolic compound content significantly increased in all walnut genotypes after inoculation by bacterial pathogens. Additionally, Zhang et al. (2022) reported a significant growth in flavonoid and phenolic compounds in walnut infected by bacterial pathogen compared to control plants, aligning with our findings. This study represents the initial comprehensive demonstration of how phenolic compounds, flavonoid content, and lignin contribution to the basal resistance of walnuts to D. necatrix. Based on the results obtained, we can conclude that the increased level of pathogenicity of D. necatrix influenced the reduction of GPX plus PPO activity and the distribution of phenolic compounds and flavonoids in this pathosystem, ultimately leading to the manifestation of the symptoms of white root rot.
Conclusions
These observations provided new insights into the crucial role of defence enzyme activity and the production of compounds related to the phenylpropanoid pathway as defence mechanisms involved in the basal resistance of walnuts to D. necatrix. Therefore, our knowledge on the defence mechanisms involved in the basal resistance as well as on the identification of virulence factors of D. necatrix, in particular the extracellular hydrolytic enzymes, and their relationship with virulence and aggressiveness, can be useful in breeding programs which would lead to the introduction of walnut genotypes with increased immunity to many pathogens.
This research was funded by the Seed and Plant Certification and Registration Institute (SPCRI), Iran (Grant number: 2-08-08-001-020013).
Conflict of interest
The authors declare that they have no conflict of interest.
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