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Article

Infection Biology of Stagonosporopsis cucurbitacearum in Watermelon and Defence Responses in the Host

by
Nguyen Thi Thu Nga
1,2,
Eigil de Neergaard
1 and
Hans Jørgen Lyngs Jørgensen
1,*
1
Department of Plant and Environmental Sciences and Copenhagen Plant Science Centre, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
2
Department of Plant Protection, College of Agriculture, Can Tho University, Can Tho City 90000, Vietnam
*
Author to whom correspondence should be addressed.
Agriculture 2024, 14(3), 380; https://doi.org/10.3390/agriculture14030380
Submission received: 8 January 2024 / Revised: 15 February 2024 / Accepted: 24 February 2024 / Published: 27 February 2024
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
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Abstract

:
Infection biology and defence responses of watermelon attacked by Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae) were studied in two genotypes, accessions PI189225 (moderately resistant) and 232-0125/B (susceptible). On intact leaf surfaces, spores started to germinate 14 h after inoculation (hai) with one to three germ tubes, which subsequently developed and formed appressoria. Invasion of the host tissue started at 20 hai by direct penetration from appressoria or occasionally indirectly through stomata. In the susceptible accession, a significantly higher number of direct penetrations were observed than in the moderately resistant. After invasion, hyphal colonisation was restricted in the intercellular spaces in the moderately resistant accession, whereas they developed extensively, causing tissue decay, in the susceptible accession. Macroscopic symptoms were seen in leaves of the moderately resistant accession as small and dry lesions, whereas big, water-soaked lesions developed on the susceptible accession within 48 hai. Investigations of the defence responses of the two accessions showed accumulation of H2O2 at penetration sites beneath appressoria in the moderately resistant, but to a lesser extent in the susceptible accession. Such H2O2 accumulation correlated with a reduction in penetration frequency and a lower level of hyphal growth after infection in the moderately resistant accession. There was a rapid and early increase in total peroxidase as well as β-1,3-glucanase activity in the moderately resistant compared to the susceptible accession. These results indicate that fungal penetration and development in watermelon are inhibited by a consorted action of different responses including accumulation of H2O2, peroxidase and β-1,3-glucanase.

1. Introduction

Gummy stem blight, caused by Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae), is a widespread and destructive disease on watermelon and other species in Cucurbitaceae. The pathogen infects all parts of the plant such as leaves, stems, fruit [1] and roots [2]. Under favourable conditions, the disease causes serious losses in watermelon in the field as well as after harvest [3,4,5]. In Vietnam, the disease is serious and complete yield loss may occur in the rainy season if spraying with fungicides is not carried out [3]. Control of the disease is a problem for several reasons. Thus, epidemics of the disease develop rapidly when humidity is high [3] and the pathogen can survive in infected host debris and soil for at least two years [1,6]. In addition to sanitary precautions and crop rotations, application of fungicides is the main method for control [3,4]. However, fungicide resistance has been observed [7,8] and no accessions with high levels of resistance are available [9]. Nevertheless, use of resistant accessions may be the most environmentally friendly strategy to limit yield losses caused by the disease and search for resistance and breeding are ongoing [9,10,11]. A prerequisite for optimal use of the available sources of resistance is an understanding of the infection biology of the pathogen as well as the defence responses in the host.
The infection biology of S. cucurbitacearum in watermelon has not been studied in much detail. An early study reported that in cotyledons of watermelon, the pathogen penetrates directly through the cuticle and intact cell walls, but no detailed descriptions of the infection were given [12]. In cotyledons of cucumber, the fungus has also been reported to penetrate directly through the intact cuticle, through damaged trichomes or through hydathodes connected to the midrib [12,13]. Direct penetration has furthermore been reported in watermelon leaves protected against disease by application of the bacterial strain Pseudomonas aeruginosa 231-1 [14].
In the flower of cucumber, the pathogen may invade through the nectarial pores and invasion through intact stigma and style tissue leads to internal fruit rot [15]. If infection occurs through wounds on the surfaces of fruit, a completely different development take place, leading to external fruit rot, characterised by extensive tissue decay [15]. Similarly, stem invasion originating from wounds results in stem decay (stem blight) and occasionally plant death [16].
Studies of direct defence responses of cucurbit plants to infection by S. cucurbitacearum have only been performed to a limited extent and apparently, there are very few reports on defence responses in watermelon. Nga et al. [14] studied resistance induced by Pseudomonas aeruginosa 231-1 in watermelon and found that protection correlated with hydrogen peroxide (H2O2) accumulation, increased peroxidase activity and occurrence of new peroxidase isoforms. In cucumber, the development of lignified cell layers in the fruit was found to play a role in resistance to S. cucurbitacearum causing internal fruit rot [15]. RNAseq studies of cucurbit plants infected by S. cucurbitacearum [17,18] also addressed events during infection, but not specific defence reactions.
Studies of induced resistance in muskmelon using Trichoderma asperelloides PSU-P1 revealed upregulation of the defence-related enzymes chitinase and β-1,3-glucanase [19,20], as well as peroxidase and polyphenol oxidase [20]. Another study in muskmelon used a crude oligosaccharide from the fungus Talaromyces verruculosus AB2 to control S. cucurbitacearum [21]. The authors implicated lignin, callose, H2O2, phenolics and the hypersensitive response in the defence together with elevated levels of the enzymes phenylalanine ammonia lyase, peroxidase, polyphenol oxidase and lipoxygenase.
Studying the quantitative and temporal expression of defence reactions and linking them to the infection of the pathogen gives a strong basis for understanding defence and ultimately resistance. Quantitative microscopy of infection progress can help to determine directly where and when pathogen growth is arrested and correlations to defence responses will make it clear whether these can explain arrest of pathogen growth.
The present study was conducted to investigate the infection biology of S. cucurbitacearum in a susceptible and a moderately resistant accession of watermelon. Furthermore, we investigated whether accumulation of H2O2, peroxidase and β-1,3-glucanase were involved in the defence of watermelon against the pathogen.

2. Materials and Methods

2.1. Plants

Two accessions of watermelon (Citrullus spp.) were used in the experiments: the moderately resistant Citrullus amarus, accession PI189225 from USDA, ARS, National Genetic Resources Program (originally collected from Zaire) and the susceptible Citrullus lanatus, accession 232-0125/B, from India. Seeds were sown in square pots (10 × 10 cm) containing the plant growth medium (peat) Pindstrup Substrate (Pindstrup Mosebrug A/S, Ryomgaard, Denmark), with two plants in each. Plants were grown in a growth chamber with alternating cycles of 12 h light and 12 h darkness. Light was supplied by fluorescent tubes (Osram L 36W/11-860 Lumilux plus Eco Daylight, Osram GmbH, Augsburg, Germany, 200 µE m−2 s−1). Temperature and relative humidity (RH) were 25 °C/50–60% RH and 20 °C/80–90% RH in light and darkness, respectively.

2.2. Inoculum of Stagonosporopsis cucurbitacearum and Inoculation

S. cucurbitacearum isolate I16 was isolated from an infected watermelon leaf collected from Can Tho Province, Vietnam in 2003. For production of inoculum, the fungus was cultured on sterilised potato cubes in 100 mL conical flasks and incubated as above for 5–7 days, as earlier described [15,16]. Spores were harvested by lightly scraping the surface of the potato cubes with a spatula and then washing them with sterilised distilled water. The spore suspension was filtered through four layers of cheesecloth, the concentration of inoculum was determined by a haemocytometer and adjusted to 105 spores/mL with sterile distilled water.

2.3. Leaf Infection

When the first true leaves were expanded (ca. 15 days after sowing), they were inoculated by atomising a spore suspension onto the leaf surface until run-off. After inoculation, plants were sealed in plastic bags to secure 100% humidity for 48 h in darkness. Subsequently, the bags were opened and the plants were placed at normal light regime again. Four days after inoculation, disease was assessed by visually recording percentage leaf area covered with symptoms as earlier described [14]. Fourteen plants of each accession were scored.

2.4. Stem Infection

Stem infection was studied on accessions PI189225 and 232-0125/B under greenhouse conditions. Before inoculation, 15-day-old seedlings were wounded by cutting off the first true leaf, leaving 1 cm of the petiole [22]. Stem inoculations were conducted by spraying S. cucurbitacearum at the wounded sites until run-off. Subsequently, inoculated plants were incubated for 48 h with 100 % humidity in darkness in the same growth chamber as before. Assessment of the disease was performed by measuring the length of stem lesions at 4 and 9 days after inoculation (dai). Twenty-four plants of each accession were scored.

2.5. Infection Biology and Accumulation of H2O2

For microscopy of the infection biology of S. cucurbitacearum, the inoculated first leaves were harvested at 14, 20 and 44 h after inoculation (hai). Leaves were cleared using a mixture of absolute ethanol: glacial acetic, 3:1 (v/v) followed by observation by light microscopy after staining with 0.1% (w/v) Evans Blue in lactoglycerol as described previously [23]. Four leaves were observed for each time point in each accession. At the early time points 14 and 20 hai, 50 randomly germinated spores were recorded on each leaf (total 200 germinated spores per accession per time point). For each germinated spore, it was recorded whether the spore had formed appressoria and whether penetration had taken place. At 44 hai, 50 random appressoria were recorded on each leaf (200 appressoria per accession per time point). For each appressorium, it was recorded whether it caused successful penetration and whether the pathogen after penetration ramified in the intercellular spaces.
The accumulation of H2O2 was studied by staining with 0.1 % (w/v) 3,3’-diaminobenzidine solution (DAB, Sigma, Søborg, Denmark) as described previously [23,24]. The inoculated leaves were harvested at 6, 12 and 36 hai and dipped immediately in DAB solution for 8 h. Subsequently, leaves were cleared using the same procedure as above, corresponding to 14, 20 and 44 hai, respectively, and observed by light microscopy after staining with 0.1 % (w/v) Evans Blue in lactoglycerol. At the early time point 14 hai, 50 randomly germinated spores were recorded on each leaf (200 spores per accession per time point). At 44 hai, 50 randomly selected appressoria were recorded on each leaf (200 appressoria per accession per time point). Reddish-brown colour (DAB-positive staining reaction for H2O2) was recorded for each germinated spore, which formed an appressorium. Furthermore, it was recorded whether positive reactions were observed at sites with successful penetration, originating from an appressorium and whether such penetrations were accompanied by ramifying hyphae with positive DAB-reaction in the intercellular spaces.

2.6. Protein Extraction for Studies of Peroxidase and β-1,3-Glucanase

Leaf samples were harvested at 0, 6, 12, 24, 36, 48, 72 and 96 hai, immediately frozen in liquid nitrogen and stored at −80 °C. The samples were ground in liquid nitrogen and proteins were extracted in the extraction buffers 0.1 M potassium phosphate (pH 7.0) for peroxidase and 0.05 M sodium acetate (pH 5.2) for β-1,3-glucanase. Protein concentration in the extract was determined using the Bradford method [25], using bovine serum albumin (Sigma, Søborg, Denmark) as standard.

2.7. Peroxidase

Peroxidase activity was determined as described by Kapchina-Toteva et al. [26]. The reaction mixture consisted of 0.25% (v/v) guaiacol (Sigma, Søborg, Denmark) and 10 mM H2O2 (Sigma, Søborg, Denmark) dissolved in 0.1 M potassium phosphate (pH 7.0). Enzyme activity was measured by a plate reader (Spectra MAX, 190, SoftMax Pro, Molecular Devices, San Jose, CA, USA). Protein extract (1–5 µL) was added in each well of the 96-well plate, and the reaction was initiated by adding 255 µL reaction mixture. The increase in absorbance (470 nm) was followed for 3 min with 10 s intervals. The rates were determined from the linear phase of the slope and the specific activities calculated. The increase in absorbance at 470 nm results from the formation of the coloured product tetraguaiacol. Specific peroxidase activity was calculated in µmol guaiacol dehydrogenated product (GDHP) mg protein−1 s−1, using the molar extinction coefficient of GDHP (ε = 26.6 mM−1cm−1).

2.8. β-1,3-Glucanase

β-1,3-glucanase activity was determined following previously described methods [27,28], with glucose (Sigma, Søborg, Denmark) as standard. The substrate was 0.1% (w/v) laminarin (Sigma, Søborg, Denmark) in 50 mM sodium acetate buffer (pH 5.2). The reaction was carried out by adding 225 µL laminarin and 25 µL enzyme extract into a microcentrifuge tube, which was incubated for 15 min at 37 °C. The reaction was stopped by adding 250 µL dinitrosalicylic reagent and boiling the samples in a water bath for 10 min. Finally, the absorbance was measured at 540 nm in a plate reader (Spectra MAX, 190, SoftMax Pro, Molecular Devices, San Jose, CA, USA). Specific activity is expressed as µmol glucose released mg protein−1 min−1.

2.9. Native PAGE for Detection of Peroxidase Isoforms

Detection of isoforms of peroxidase in native PAGE followed the methods of [14,29]. Forty-microgram protein was loaded onto a 10% polyacrylamide gel. A basic gel was used for detection of acidic isoforms. After electrophoresis, peroxidase isoforms were stained by rinsing the gel in a solution containing 10 mM H2O2 and 0.2% (w/v) benzidine (Sigma, Søborg, Denmark) dissolved in 0.1% (v/v) glacial acetic acid, which was subsequently filtered through cotton. The gel was rinsed in the solution with continuous shaking for approximately 20 min until all the isoforms appeared. The reaction was stopped by adding distilled water and the gel was washed several times to remove excess stain.

2.10. Experimental Design and Statistical Analysis of Data

Data from studies of infection biology and H2O2 accumulation represent discrete variables and were therefore assumed to follow a binomial distribution. They were analysed by logistic regression as described previously [23,30]. Data for leaf infection, length of stem lesions and enzyme activities represent continuous variables and were analysed by analysis of variance assuming a normal distribution. Variances were stabilised by appropriate transformations when necessary. All data were analysed by PC-SAS (release 9.4; SAS Institute, Cary, NC, USA) and hypotheses rejected at p < 0.05.

3. Results

3.1. Disease Development

In the susceptible accession 232-0125/B, leaf symptoms appeared 2 days after inoculation (dai) as water-soaked lesions, covering 58.9% of the inoculated area by 4 dai (Figure 1A, Table 1). In the moderately resistant PI189225, symptoms also started to appear at 2 dai as small, dry and necrotic lesions, but at 4 dai, only 4.0% of leaf area was infected (Figure 1B, Table 1), which was significantly lower compared to the susceptible accession.
In stem infections through wounds, symptoms started at 2 dai as water-soaked lesions in both accessions. Infection started at the wound sites and rapidly developed upwards and downwards. Stem lesions in PI189225 were significantly shorter than in 232-0125/B at 4 and 9 dai (Table 1). At 9 dai, severe infection was seen in 232-0125/B, leading to plant collapse, whereas in PI189225, most plants were still alive and standing up (Figure 2C,D). Formation of pycnidia was seen in the lesions at 4 dai in 232-0125/B, whereas this only occurred at 7 dai in PI189225.

3.2. Infection Biology and Accumulation of H2O2 at Penetration Sites in Leaves

Spores were observed germinating at 14 hai with one to three germ tubes, which formed appressoria (Figure 2A,D). There was no significant difference in the percentage of spores forming appressoria between the two accessions at 14 and 20 hai (Table 2). From 20 hai, penetration started to occur from the appressoria. Penetration took place directly through the intact leaf surface or very rarely through stomata (a very low percentage of the penetrations), with most penetrations occurring through anticlinal cell walls or between the guard cells of stomata (Figure 2B,D,E). At 20 hai, penetration had already occurred in 232-0125/B, whereas it took place later in PI189225. The percentage appressoria causing penetration was significantly higher in 232-0125/B than in PI189225 at 20 and 44 hai (9.8% and 22.5% in 232-0125/B and 0.0% and 15.5% in PI189225, respectively). After successful penetration, S. cucurbitacearum started to ramify in the intercellular spaces and break down tissues. There was a significantly higher number of appressoria with successful penetration and intercellular growth in 232-0125/B than in PI189225 at 44 hai (8.0% and 4.5%, respectively) (Figure 2C,F). Macroscopic symptoms on leaves were seen in both accessions at 44 hai, with small dry lesions in PI189225 and large water-soaked lesions in 232-0125/B.
DAB staining of leaves infected by S. cucurbitacearum showed that H2O2 accumulation did not take place below any of the appressoria at 14 hai in 232-0125/B, whereas 54.2 % of the appressoria had H2O2 accumulation in PI189225 (Table 2, Figure 2A,D). At 20 and 44 hai, the percentage of appressoria with H2O2 accumulation increased in 232-0125/B and reached almost the same level as in PI189225 (39.8 and 33.0% at 20 hai and 26.0 and 28.0% at 44 hai, respectively) (Table 2, Figure 2B,E).

3.3. Peroxidase Activity

The total peroxidase activity in the control (only treated with water) of both accessions did not increase during the time course study (Figure 3). Inoculation with the pathogen resulted in a significant increase in peroxidase activity from 24 to 36 hai in both accessions. In PI189225 inoculated with pathogen, peroxidase activity increased rapidly and reached significantly higher levels than the control treated with water at 36 hai. In 232-0125/B, peroxidase activity also increased from 24 hai, but this was not significantly higher than in the control at 36 hai (Figure 3).
During the later time points 48 to 72 hai, the peroxidase activity increased rapidly and reached the highest level at 72 hai in both accessions inoculated with the pathogen (Figure 3). This was accompanied by symptom expression and external hyphal colonisation. There was a significantly higher level of peroxidase activity in accession 232-0125/B than in accession PI189225 at 72 hai.

3.4. Peroxidase Isoforms

Basic native PAGE was used for detection of acidic isoforms of peroxidase (Figure 4). In PI189225 treated with water (Figure 4A), an acidic 45 kDa isoform was more prominent than in 232-0125/B (Figure 4C). After inoculation with the pathogen, this isoform rapidly accumulated in PI189225 at 6 to 36 hai (Figure 4B). This was not seen in 232-0125/B inoculated with the pathogen (Figure 4D), where the isoform started to increase only from 48 hai.

3.5. β-1,3-Glucanase Activity

During the early stages of infection (0 to 48 hai), β-1,3-glucanase activity increased significantly at 12 and 24 hai in PI189225 compared to 0 hai in both the control treated with water and in plants inoculated with pathogen (Figure 5). However, the activity was not significantly different between these two treatments. By contrast, in 232-0125/B, there was no increased activity of β-1,3-glucanase at the early time points in the control plants treated with water or inoculated with pathogen (Figure 5).
During the late stage of infection (48 to 96 hai), β-1,3-glucanase activity started to increase early from 48 hai in PI189225 inoculated with the pathogen and reached the highest level at 96 hai, which was significantly higher than in the control treated with water (Figure 5). In 232-0125/B, activity started to increase only from 72 hai and it was significantly higher for the inoculated plants than in the control treated with water at 96 hai (Figure 5). Comparison of PI189225 and 232-0125/B showed that there was no significant difference between the water controls whereas the activity in the inoculated plants started to increase earlier (48 hai) in PI189225 than in 232-0125/B and was significantly higher at 72 and 96 hai (Figure 5).

4. Discussion

Accession PI189225 showed significantly less leaf and stem infection compared to the susceptible accession 232-0125/B. PI189225 is considered as resistant and has served for breeding purposes as a source of resistance [6,9,10,11]. However, even though it is more resistant than 232-0125/B, severe stem symptoms were recorded in the present investigation, confirming that accession PI189225 is only moderately resistant to gummy stem blight. Lesions on stems grew in both directions, i.e., upwards and downwards. This indicates that fungal spread takes place by hyphal growth within the stem tissue. Stem infection was achieved by cutting the petiole of the first true leaf and applying the pathogen to the wounds. Development of stem symptoms occurred earlier here than in intact plants, indicating that wounding is an important factor influencing disease development.
In watermelon leaves, S. cucurbitacearum was found to penetrate directly through the intact surface or indirectly through the stomata. In previous studies of infection of watermelon leaves, it was reported that S. cucurbitacearum was able to penetrate directly through the cuticle, whereas penetration through the stomata was not reported [12]. In cucumber, the fungus can penetrate the cuticle directly in cotyledons, but most often penetration occurs through hydathodes and injured trichomes [12]. In flower parts, S. cucurbitacearum colonises host tissues through the intact stigma and style, leading to internal fruit rot [15]. On the fruit and stems of cucumber, infection through wounds leads to extensive tissue decay, causing outer fruit rot or stem blight [16]. Collectively, these observations show that S. cucurbitacearum is able to invade the host by several different ways.
We found that H2O2 accumulation occurred at the penetration sites of S. cucurbitacearum in watermelon leaves. There was a significantly higher percentage of appressoria with H2O2 accumulation at 14 hai in the moderately resistant than in the susceptible accession and this correlated with a significant reduction in penetration at 20 and 44 hai. Together, these results indicate that early accumulation of H2O2 may be related to a reduction in the penetration and inhibition of the extent of fungal colonisation.
The oxidative burst and the accumulation of PR-proteins have been reported as important in plant resistance [31,32,33,34]. The oxidative burst is a rapid increase in the level of certain reactive oxygen species in plants when attacked by pathogens. It is considered as part of the early defence against pathogen attack [31,32,35,36]. H2O2 is a relatively stable reactive oxygen species, produced by many plants as a response to pathogen attack [36]. The role of H2O2 in defence has been reported from many host–pathogen interactions [23,30,35,37,38,39]. H2O2 produced at the penetration sites can directly inhibit pathogen penetration [23,24,30,37,39]. In addition, H2O2 participates in oxidative cross-linking of proteins in the cell walls, lignification and furthermore, H2O2 may also serve as a signalling compound activating defence responses [32,35,40]. There has apparently been no research on the involvement of H2O2 in defence in cucurbits.
The accumulation of H2O2 at penetration sites is important in the defence against biotrophic pathogens such as Blumeria hordei infecting barley, Uromyces vignae infecting pea and Golovinomyces cichoracearum infecting cowpea [24,37]. H2O2 has also been found to inhibit hemibiotrophic pathogens during the initial stage of infection when the pathogens are in their biotrophic stage. Examples are Bipolaris sorokiniana infecting barley, Zymoseptoria tritici infecting wheat and Bipolaris oryzae infecting rice [23,30,41]. During the late stages of infection, when these pathogens change to necrotrophic growth, H2O2 accumulation could still be harmful [42], but in some cases, it is considered beneficial for pathogens [41]. Thus, for necrotrophic pathogens like Botrytis cinerea, it has been suggested that H2O2 accumulation is beneficial for the infection and colonisation of the pathogen [43,44]. However, in contrast to this suggestion, it has also been observed that accumulation of H2O2 can inhibit the penetration of Botrytis cinerea in tomato and bean during early stages of infection [45,46].
Traditionally, S. cucurbitacearum has been considered a facultative necrotroph [47]. The present study showed that H2O2 appears to inhibit the pathogen during the initial penetration and in addition, we saw that S. cucurbitacearum can penetrate directly through intact tissue. Furthermore, the fact that it can grow intercellularly without causing damage to the plant tissues for some times as observed in flower infection [15] suggests that the fungus may have an initial biotrophic phase. The same growth pattern was observed for the hemibiotroph pathogens Bipolaris sorokiniana infecting wheat/barley [48] and for Bipolaris oryzae infecting rice [30]. Therefore, we suggest that S. cucurbitacearum should be considered a hemibiotroph, although further research is required to establish how the biotrophic growth changes into necrotrophic behaviour.
In Table 2, the difference between the fungal developmental steps and host responses at 44 hai does not appear to be able to explain the dramatic difference observed in disease severity between the two accessions. This is most likely because resistance in the moderately resistant accession is expressed at several stages during infection and colonisation, whereas Table 2 only addresses the first days after infection. Thus, the early inhibition is accompanied by changes in activities of peroxidase and β-1,3-glucanase as well as peroxidase isoform pattern changes. Additional defence responses are also likely activated.
At 36 hai, peroxidase activity was significantly higher in accession PI189225 inoculated with S. cucurbitacearum compared to the water-treated control, whereas in accession 232-0125/B inoculated with the pathogen, peroxidase activity increased only slightly and was not significantly different from the control treated with water. Peroxidases are important enzymes involved in many defence responses. Firstly, peroxidases are involved in generation and degradation of H2O2 [35,36,49]. Peroxidases also participate in lignification, suberisation, and in oxidative protein cross-linking in cell walls [50,51].
The increase in peroxidase activity correlated with a higher accumulation of H2O2 at infection sites in accession PI189225 compared to accession 232-0125/B at 44 hai. In addition, there was an early and prominent accumulation of an acidic 45 kDa isoform of peroxidase in accession PI189225 already at 6 hai compared to accession 232-0125/B where this isoform was only seen at 36 hai. The increase in peroxidase activity and early accumulation of an acidic 45 kDa peroxidase isoform correlated with inhibition of fungal penetration.
Increased activity and accumulation of different isozymes of peroxidase, correlating with resistance have been reported in different plant–pathogen interactions, e.g., in pearl millet infected by Sclerospora graminicola [52] and in wheat infected by Zymoseptoria tritici [23]. In cucurbit plants, Nga et al. [14] studied watermelon protected against gummy stem blight by application of Pseudomonas aeruginosa 231-1. They found that protection was accompanied by increased peroxidase activity as well as an increased accumulation of a 45-kDa acidic peroxidase isoform. The same isoform was found to accumulate in the current study, especially in the moderately resistant accession, thus further corroborating a role in defence against the pathogen. Several other acidic and basic isoforms accumulated and likewise, peroxidase isoforms of varying sizes have been implicated in resistance of cucurbit plants against different pathogens [53,54,55,56,57]. Further research is needed to determine the precise role of the various isoforms in defence in cucurbit plants.
In the late stages of infection (72 hai), peroxidase activity increased rapidly in accession 232-0125/B inoculated with S. cucurbitacearum and became significantly higher than in accession PI189225. This increase correlated with rapid symptom development in the susceptible accession. Similarly, in wheat infected with Zymoseptoria tritici, peroxidase activity was also very high in a susceptible accession at the late stage of the infection, correlating with fungal proliferation [23]. This rapid increase in peroxidase activity in the susceptible watermelon accession could be a late defence response, occurring too late to stop the pathogen. Thus, the pathogen already established and proliferated to an extent where mounting of defence would not have a major impact on it. Another possibility could be that the increase reflects accumulation of ascorbate peroxidase as seen in sunflower [58]. Total peroxidase activity includes both peroxidases that generate and degrade H2O2. During the late stage of infection, high amounts of H2O2 were produced during pathogen colonisation and this will be toxic to the host [59]. Therefore, the plants will produce higher amounts of ascorbate peroxidase to remove H2O2, leading to an increase in total peroxidase activity, correlating with severe infection. In the present study, significantly higher ascorbate peroxidase activity was seen in the susceptible than in the resistant accession at 72 hai. Future studies should address the occurrence of specific peroxidases as well as other antioxidants to elucidate their roles in defence in watermelon against S. cucurbitacearum.
We found that β-1,3-glucanase activity increased when symptoms of disease appeared. In accession PI189225, the activity of β-1,3-glucanase increased to a high level earlier than in accession 232-0125/B. The increase in β-1,3-glucanase activity correlated with restriction of lesion development in the moderately resistant accession. β-1,3-glucanase (PR-2) has been reported to be an important enzyme in plant defence [34,60]. The enzyme can degrade the fungal cell walls and inhibit fungal growth and, in addition, oligosaccharide fragments released by enzyme activity may serve as elicitors to activate different defence responses [28,34,60]. Early β-1,3-glucanase activity is often higher in incompatible than in compatible interactions, for example, in wheat infected by Zymoseptoria tritici [28]. This was also observed in muskmelon infected by Fusarium oxysporum f.sp. melonis [61], and β-1,3-glucanase activity was also found to be associated with resistance of squash and pumpkin against Gloeosporium orbiculare [62]. Likewise, induction of resistance using Trichoderma asperelloides PSU-P1 as the inducer in muskmelon against S. cucurbitacearum also involved elevated transcript levels as well as increased enzyme activity of β-1,3-glucanase [19,20].

5. Conclusions

Early accumulation of H2O2 at penetration sites, early increase in peroxidase activity and accumulation of an acidic 45 kDa peroxidase isoform combined with higher activity of β-1,3-glucanase appear to be important factors involved in the defence of the moderately resistant accession PI189225 of watermelon against S. cucurbitacearum.

Author Contributions

Conceptualization, N.T.T.N., E.d.N. and H.J.L.J.; methodology, N.T.T.N., E.d.N. and H.J.L.J.; formal analysis, N.T.T.N., E.d.N. and H.J.L.J.; investigation, N.T.T.N., E.d.N. and H.J.L.J.; data curation, N.T.T.N.; writing—original draft preparation, N.T.T.N., E.d.N. and H.J.L.J.; writing—review and editing, N.T.T.N., E.d.N. and H.J.L.J.; visualisation, N.T.T.N. and H.J.L.J.; supervision, E.d.N. and H.J.L.J.; project administration, E.d.N. and H.J.L.J.; funding acquisition, E.d.N. and H.J.L.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by a grant from the DANIDA-ENRECA (Enhancement of Research Capacity) project: “Systemic Acquired Resistance—an eco-friendly strategy for managing diseases in rice and pearl millet” financed by the Danish Ministry of Foreign Affairs.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All data are contained within the article.

Acknowledgments

The authors would like to thank Todd C. Wehner, North Carolina State University, USA for providing accession PI189225 and Shekar H. Shetty, Sharathchandra R. G., Hindumathi C. K. and Geetha N. P., Department of Biotechnology, Faculty of Science and Technology, University of Mysore, India, for their guidance on enzyme assays and protein electrophoresis.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Figure 1. Symptoms of Stagonosporopsis cucurbitacearum on the first true leaf of watermelon at 4 days after inoculation in (A) the susceptible accession 232-0125/B and (B) the moderately resistant accession PI189225. Symptoms of Stagonosporopsis cucurbitacearum on the stem of watermelon at 9 days after inoculation in (C) the susceptible accession 232-0125/B and (D) the moderately resistant accession PI189225.
Figure 1. Symptoms of Stagonosporopsis cucurbitacearum on the first true leaf of watermelon at 4 days after inoculation in (A) the susceptible accession 232-0125/B and (B) the moderately resistant accession PI189225. Symptoms of Stagonosporopsis cucurbitacearum on the stem of watermelon at 9 days after inoculation in (C) the susceptible accession 232-0125/B and (D) the moderately resistant accession PI189225.
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Figure 2. Time course of infection of Stagonosporopsis cucurbitacearum and the accumulation of H2O2 at penetration sites in the susceptible accession 232-0125/B of watermelon (A–C) and the moderately resistant accession PI189225 (D–F). (A) Spore germinates with two germ tubes at 20 hai in accession 232-0125/B (arrows). (B) Appressorium (arrow) with direct penetration attempt through a guard cell and faint accumulation of H2O2 (DAB staining) at 32 hai in accession 232-0125/B. (C) Fungal growth in the intercellular spaces without accumulation of H2O2 at the penetration site at 44 hai in accession 232-0125/B. (D) Appressorium with attempted penetration directly through the cuticle and H2O2 accumulation at the penetration site at 20 hai in accession PI189225 (arrow). (E) Appressorium with successful penetration and H2O2 accumulation at the penetration site at 32 hai in accession PI189225. (F) Fungal growth in the intercellular spaces with prominent accumulation of H2O2 at the penetration site at 44 hai in accession PI189225.
Figure 2. Time course of infection of Stagonosporopsis cucurbitacearum and the accumulation of H2O2 at penetration sites in the susceptible accession 232-0125/B of watermelon (A–C) and the moderately resistant accession PI189225 (D–F). (A) Spore germinates with two germ tubes at 20 hai in accession 232-0125/B (arrows). (B) Appressorium (arrow) with direct penetration attempt through a guard cell and faint accumulation of H2O2 (DAB staining) at 32 hai in accession 232-0125/B. (C) Fungal growth in the intercellular spaces without accumulation of H2O2 at the penetration site at 44 hai in accession 232-0125/B. (D) Appressorium with attempted penetration directly through the cuticle and H2O2 accumulation at the penetration site at 20 hai in accession PI189225 (arrow). (E) Appressorium with successful penetration and H2O2 accumulation at the penetration site at 32 hai in accession PI189225. (F) Fungal growth in the intercellular spaces with prominent accumulation of H2O2 at the penetration site at 44 hai in accession PI189225.
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Figure 3. Total peroxidase activity (µmol guaiacol dehydrogenated product (GDHP)/mg protein/second) in watermelon leaves of the moderately resistant accession PI189225 and the susceptible accession 232-0125/B after inoculation with Stagonosporopsis cucurbitacearum or application of water. Error bars represent standard error of the mean.
Figure 3. Total peroxidase activity (µmol guaiacol dehydrogenated product (GDHP)/mg protein/second) in watermelon leaves of the moderately resistant accession PI189225 and the susceptible accession 232-0125/B after inoculation with Stagonosporopsis cucurbitacearum or application of water. Error bars represent standard error of the mean.
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Figure 4. Basic 10% native PAGE showing acidic peroxidase isozymes in leaves of the moderately resistant accession PI189225 (A,B) and the susceptible accession 232-0125/B (C,D) of watermelon after inoculation with Stagonosporopsis cucurbitacearum or treatment with water. (A) Accession PI189225—treated with water. (B) Accession PI189225—inoculated with S. cucurbitacearum. (C) Accession 232-0125/B—treated with water. (D) Accession 232-0125/B—inoculated with S. cucurbitacearum.
Figure 4. Basic 10% native PAGE showing acidic peroxidase isozymes in leaves of the moderately resistant accession PI189225 (A,B) and the susceptible accession 232-0125/B (C,D) of watermelon after inoculation with Stagonosporopsis cucurbitacearum or treatment with water. (A) Accession PI189225—treated with water. (B) Accession PI189225—inoculated with S. cucurbitacearum. (C) Accession 232-0125/B—treated with water. (D) Accession 232-0125/B—inoculated with S. cucurbitacearum.
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Figure 5. β-1,3-glucanase activity (µmol glucose released/mg protein/min) in watermelon leaves of the moderately resistant accession PI189225 and the susceptible accession 232-0125/B after inoculation with Stagonosporopsis cucurbitacearum or treatment with water. Error bars represent standard error of the mean.
Figure 5. β-1,3-glucanase activity (µmol glucose released/mg protein/min) in watermelon leaves of the moderately resistant accession PI189225 and the susceptible accession 232-0125/B after inoculation with Stagonosporopsis cucurbitacearum or treatment with water. Error bars represent standard error of the mean.
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Table 1. Severity of gummy stem blight on leaves and length of stem lesions on watermelon of the moderately resistant accession PI189225 and the susceptible accession 232-0125/B at 4 days after inoculation with Stagonosporopsis cucurbitacearum.
Table 1. Severity of gummy stem blight on leaves and length of stem lesions on watermelon of the moderately resistant accession PI189225 and the susceptible accession 232-0125/B at 4 days after inoculation with Stagonosporopsis cucurbitacearum.
Accession Percentage Leaf Area with Symptoms 4 daiLength of Stem Lesion (mm)
4 dai9 dai
PI1892254.0 2.96.7
232-0125/B58.9 4.910.3
LSD9522.60.70.8
p-value0.00020.00010.0001
dai: days after inoculation.
Table 2. Infection biology of Stagonosporopsis cucurbitacearum and accumulation of H2O2 at penetration sites in leaves of the moderately resistant accession PI189225 (R) and the susceptible accession 232-0125/B (S) of watermelon.
Table 2. Infection biology of Stagonosporopsis cucurbitacearum and accumulation of H2O2 at penetration sites in leaves of the moderately resistant accession PI189225 (R) and the susceptible accession 232-0125/B (S) of watermelon.
Percentage of Time after Inoculation with Stagonosporopsis cucurbitacearum
14 hai20 hai44 hai
RS RS RS
spores forming appressoria4.65.0ns13.817.1ns- b- b
appressoria causing penetration- a- a-0.09.8**15.522.5*
appressoria with successful penetrations with tissue colonisation - a- a-- a- a-4.58.0***
appressoria with H2O2 accumulation54.20.0***39.833.0ns26.028.0ns
The number of asterisks indicates the degree of significance: *: significant at 0.01 < p < 0.05; **: significant at 0.001 < p < 0.01; ***: significant at p < 0.001, ns: non-significant difference (p > 0.05). -a: the event did not occur at the observed time point. -b: data not recorded.
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Nga, N.T.T.; de Neergaard, E.; Jørgensen, H.J.L. Infection Biology of Stagonosporopsis cucurbitacearum in Watermelon and Defence Responses in the Host. Agriculture 2024, 14, 380. https://doi.org/10.3390/agriculture14030380

AMA Style

Nga NTT, de Neergaard E, Jørgensen HJL. Infection Biology of Stagonosporopsis cucurbitacearum in Watermelon and Defence Responses in the Host. Agriculture. 2024; 14(3):380. https://doi.org/10.3390/agriculture14030380

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Nga, Nguyen Thi Thu, Eigil de Neergaard, and Hans Jørgen Lyngs Jørgensen. 2024. "Infection Biology of Stagonosporopsis cucurbitacearum in Watermelon and Defence Responses in the Host" Agriculture 14, no. 3: 380. https://doi.org/10.3390/agriculture14030380

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