Inducing systemic acquired resistance (SAR) against root-knot nematode Meloidogyne javanica and evaluation of biochemical changes in cucumber root

Plant parasitic nematodes are responsible for severe yield loss, reduced crop production, as well as cause damage to economically important crops (Schleker et al., 2022). Root-knot nematodes are one of the most important groups of plant parasitic nematodes and cause heavy yield losses annually, causing significant damage and production loss in many cultivated plants, particularly vegetable crops (Feyisa, 2021). Root galls’ induction has been shown to restrict water and nutrient uptake, leading to wilting, mineral deficiencies, reduced plant growth, and decreased yield and plant biomass (Hallmann, 2018). In addition, Meloidogyne spp. is one of the most destructive pathogens in cucumber crops which causes damage and reduces growth and yield loss (Muther, 2020); the yield loss may reach up to 50 % (Katooli et al., 2010).

During a survey carried out in north Iraq, results showed that a high disease incidence was in cucumbers 58 % and tomatoes 33 %, which are considered the most common vegetable grown under greenhouses (Hamad et al., 2022). Surveys in Erbil province showed a wide distribution of M. javanica in cucumber fields Ami, (2010). There are also studies in Iraq to use chemicals and plant extract to control root-knot nematodes Taher (2019).

Systemic acquired resistance induced in tomatoes against root-knot nematode reduced infection criteria and improved growth indices Bakr and Hewedy (2018). Similarly, the application of SA increased some defense compounds, such as hydrogen peroxide and peroxidase activities in the root of cucumbers infected by M. javanica (Siahpoush et al., 2011). Treatment with ascorbic acid significantly enhanced plant growth parameters, including shoot and root weight and length. Moreover, compared with untreated control plants, these resistance inducers enhanced the synthesis and activity of defense enzymes, peroxidase, and phenol oxidase. Ziadi et al. (2001).

Recent investigations have shown a reduction in the nematode population of the root-knot nematodes lower number of eggs, juveniles, and galls in the presence of silicates Roldi et al. (2017). Additional studies illustrate that silicon lowers the effect of nematode on cucumber and many other vegetable crops by decreasing infection parameters Kaushik and Saini (2019).

This investigation expands upon previous studies to show: 1) the detection of inducing systemic acquired resistance (SAR) in plants by foliar applications of salicylic acid, ascorbic acid, and silicon, 2) the evaluation of SAR criteria, including peroxidase and polyphenol oxidase activities, and 3) assessment of plant vigor through growth and infection measurements.

As demonstrated in Table 1, results show the effect of salicylic acid, ascorbic acid, and silicon application on the growth criteria of cucumbers infected by M. javanica. Chlorophyll in the healthy plant was 17.17 followed by foliar and root application of salicylic acid was recorded at 14.97, which was a significantly better treatment for increasing chlorophyll percentage when compared with other treatments. The lowest rate was in infested (control) treatment 9.3 due to infection with M. javanica and without any treatment. Nematode infection affected the cucumber plant and led to weakness and less chlorophyll density in the leaves. Foliar application of salicylic acid was recorded at 13.6 followed by foliar and root application of silicon at 12.9, and root application of silicon at 12.2. Meanwhile, foliar and root application of Ascorbic acid was at 12.17. All treatments showed better chlorophyll content than infested control one.

The highest shoots fresh weight after healthy plants were foliar and root application of salicylic acid 53.7 g, followed by root application of salicylic acid 51.8 g, foliar application of salicylic acid (SA) 50.97 g, root and foliar application of silicon (Si) 48.3 g, root application of SC47.67g, root and foliar application of ascorbic acid (AA) 46.7 g then followed by other treatments and less than 9.3 g in control.

Root fresh weight in the healthy plant was 17.17 g, significant compared with infected control and other treatments which is normal results, since root-knot nematode causes damage to the roots and malformation in the root system, affecting infected cucumber plants. Root fresh weight in treatment with root and foliar application of SA was significantly better than in other 14.97 g, followed by the SA root application 14.03 g and SA foliar application 13.6 g. Shoots dry weight values were near the healthy plant in root and foliar application of SA 9.373 g followed by 9.03, 8.857, 8.153 and 8.03 g in SA root application, the SA foliar application, the Si root and foliar application, the Si root application, and then AA root and foliar application. Due to the high level of water contained in plant vegetative parts, the dry weight is a good way to estimate plant growth and is more reliable.

Root dry weight in the healthy plant was 1.089 g, followed by the SA root and foliar application 0.9507 g, the SA root application 0.8627 g, the foliar application 0.8313 g, and the Si root and foliar application 0.81 g. After the SA treatments, the Si application recorded better results than AA. Less value, 0.5837 recorded in control without any treatments M. javanica affected in malformation and growth of roots.

Shoots length also recorded an improvement in SA application compared with control treatments; SA root and foliar recorded 63.37 cm length, SA adding to root zoon 61.3 cm shoot length. Si and AA applications also have a reasonable effect in shoot length compared to the control.

Root length in all treatments was shorter than in healthy plants due to root-knot infection, which reduces the host’s root system. Foliar and root application of SA root length were 23.9 cm, significantly different and better than in the other treatments, followed by SA root application at 21.47 cm and SA foliar sprayed at 21.03 cm. The other treatments were not significantly different but still recorded better root length compared with the control 18.07 cm. Among the other treatments, Si root and foliar application were better, with a 19.9 cm root length. AA foliar spray has less effect in improving the root length.

Results of infection criteria, as illustrated in (Table 2) showed that the number of root Knot (galls) was less in SA root and foliar applications, 23 galls/root system compared with controls 37.33 galls/root system. Thus salicylic acid root and foliar application decreased the number of galls /root systems. The Si root and foliar application were at 25.67 gall/root system, followed by the SA root application and SA foliar application at 26 and 28.33, respectively. Ascorbic acid root application was recorded at 29. AA roots and foliar application at 30.67 gall/root system reduced gall number / root compared with control. Due to the wide range of the root gall index, the number is close, but they are still significantly different treatments than the control. The root-knot index was 3 in all of the following treatments: root and leaf application of SA, SA root application, SA leaf application, Si root and leaf application, Si root application, and AA root and leaf application. The leaf applications of AA and Si did not differ significantly from the control. The gall index was 4.

The number of egg mass in SA root and foliar application was 10.33. It was followed by Si root and leaf application 13, SA root application 15.33, SA leaf application 16.33, and AA root application 17.33. The AA root and foliar application added 17.67 Si to the root zone, AA foliar spray 18.33, and Si foliar spray 20.0. All treatments were recorded less egg mass/root system and significantly different from the control 26 egg mass /root system.

More nematodes/root systems were recorded in control 499.3 and less in SA root and foliar application 395.7 after healthy plant, which is zero (not infected). In the root and leaf application of Si Number of nematodes/root system was 404.3, then 415, 418.3, 418.7, 419.7, 443.7, 461.7, and 468 for each of the SA root application, SA foliar application, AA root application, AA root, and foliar application, Si root application, AA foliar and Si foliar application respectively.

All treatments reduced the number of eggs/Root system compared with the control 5984 eggs/root system. 2196 eggs/root system for SA root and foliar application, Si root and foliar application were at 2765, SA root application at 3262, SA foliar spray at 3521, and the AA root application was at 3651. The AA root and foliar application were at 3693, the Si root application at 3702, AA foliar application at 3935, and Si foliar application at 4258. The number of nema todes/pots soil in treatments used SA in both roots, and foliar part was at 477.0. A significant decrease in the number of nematodes compared with other treatments and control after the root and foliar application of Si with 584.3 nematodes/pots soil.

Salicylic acid root and SA foliar application were 711.7 and 721.3, respectively. Nematodes/soil pots were 789.3 for AA adding to root zoon 772.7, then root and foliar application with 789.3 nematodes/ pots soil. No significant differences between root applications of Si 841.0, AA foliar spray 848.7 and Si foliar spray 859.7 were found, but still reducing the number of nematodes/soil pots compared with control recorded 874.7 nematodes/pots soil. The final population densities of nematodes were close. Still, all treatments declined the final number of nematodes and reduced the effect of nematodes on cucumber root 7492 in control. A smaller final population density of root-knot nematode was recorded in the treatment with Salicylic acid root and foliar application (3068 Si root followed by 3230 foliar application).

Salicylic acid added to root zoon resulted in 3754 nematodes. SA foliar sprayed yielded 4418 nematodes, 4839 nematodes for AA root application, and 4842 for AA root and foliar application. A lesser reduction was observed with Si treatments root application; AA foliar sprayed and Si foliar sprayed 4root zoon (953, 5252, and 5573).

All treatments in this experiment significantly reduced the reproduction rate in root-knot nematodes M. javanica which is so important for decreasing the damage and effect of nematodes and reducing population densities in the soil so that the next infection will be less severe on plants. The reproduction rate in control was 7.492, and less reproduction rate resulted from foliar root application of SA (3.068), then Si root and foliar application (3.230), SA added to root zone 3.754. The foliar spray of SA also increased the resistance of the cucumber plant. The reproduction rate reduced to 4.418, then to 4.839 and 4.842 for AA root application, followed by AA foliar and root application. All infection criteria adding AA in the root zone are more effective than application for both root and foliar for Si root application (4.953), AA foliar applications (5.252), and Si foliar sprays (5.252).

Results of total phenolic substances in cucumber plant roots (Fig. 1) showed that total phenol in root of healthy plant was 1.327mg and control recorded more phenolic substances 1.923mg as cucumber root react against pathogen root-knot nematodes and all other treatment increased number of phenolic substances as defense substances.

Fig. 1

SA(R+F) recorded highest level of phenolic substances 4.019 mg and had significant effect on increments of total phenolic substances in root, then 3.44mg and 3.414 in AA(R+F) and SA(R) respectively.

Cucumber plant sprayed with SA recorded 2.644 mg and 2.435 mg for Si(R), 2.391 mg for Si(R+F), Si (F) 2.371 mg, AA(R) 2.344 and 2.204 mg for AA (F). Ascorbic acid applications were less effect treatments on phenolic substances increment on cucumber roots. Effect of treatments salicylic acid, ascorbic acid, and Silicon application on peroxidase activity (Unit. Min-1.gm-1 fw.) in cucumber root demonstrated in (Fig. 2) due to the infection by M. javanica peroxidase activity pile up from 5.77 in healthy plant to 10.33 in control and reached the pick in SA(R+F) treatment 25.38 then 22.83, 22.11 in AA(R+F) and SA(R) they are significantly different from other treatment. SA (F), Si(R), Si(R+F), Si (F), AA(R) and AA (F) where recorded 19.77, 19.16, 19.03, 18.16, 16.42 and 14.28 respectively.

Fig. 2

Results of Polyphenol oxidase activity (Unit. Min-1.gm-1 f w.) estimation as demonstrated in Figure 3 also there is reasonable increased with treatments and reached the pick in SA(R+F) 9.067 followed by AA(R+F) 8.6, SA(R) 8.167, SA(F) 7.6 then Si(R) 6.3, Si(R+F) 6 whereas foliar application of ascorbic acid and silicon were less effect and recorded 4.7 and 4.167.

Fig. 3

The salicylic acid (SA) application, especially roots and foliar represented better treatments for reducing the effect of nematode on chlorophyll. The SA played a role in defense as phytohormones have a role in photosynthesis (Hayat et al., 2010), and these results agreed with our results in this trial. Results agreed with (López-Gómez et al., 2015), who state that Chlorophyll contents mostly decline with increasing nematode population density.

Chlorophyll content measures photosynthesis activity and nitrogen content in the plant. The decrease in chlorophyll due to root-knot nematode’s influence interferes with water and nutrient transport through the infected plant (Melakeberhan et al., 1987; Kirkpatrick et al., 1991; Carneiro et al., 2002). Thus better chlorophyll content in treatment indicates that treatment reduced the nematode effect on the cucumber plant.

Salicylic acid has a role in various enzyme activities and bio-productivity, which helps improve growth criteria besides decreasing the effect of infection (Hayat et al., 2010).

The results of growth criteria corresponded with many researchers. They stated that the application of SA growth parameters could significantly enhance the dry shoot weight, root length, shoot weight and fresh root weight, and chlorophyll (Bakr et al., 2018; Mukherjee et al., 2012; Mostafanezhad et al., 2014, and Moslemi et al., 2016). New research performed by Ami and Shingaly (2020), found that SA application improves the growth criteria.

The effect of SA on infection reduction agrees with those recorded by Bakr and Hewedy (2018). Their study of systemic resistance induction in tomato against root-knot nematode by applied Salicylic acid (SA) at 50 mM and results demonstrated the highest reduction in number of galls, egg masses, females/tomato root system and number of second stage juveniles.

Reduction in infection criteria due to SA application attributed to its role as a signaling molecule involved in both reactions at infection sites and local defense and its role in the induction of systemic resistance. Although SA can serve as a long-distance messenger signaling in presence of any stress factors such as pathogen, so it’s synthesis and accumulation are necessary requirements for defense responses (Walters etal., 2013) as well as salicylic acid (SA) considers phytohormone which Salicylic acid (SA) is a key plant hormone required for establishing resistance to many pathogens is one of the most essential signal molecules have role in activator defense responses and/or inducing plant cells for response against pathogen infection (Ding & Ding, 2020). Researches also coincide with our results that silicon played important role in reducing the effect of biotic and abiotic stress in vegetable crops including root knot nematodes, researches illustrate that silicon lowers the effect of nematode on cucumber and many other vegetable crops by decreasing infection parameters. (Kaushik & Saini, 2019). Reduction in the nematode population was observed in the presence of Si under greenhouse conditions (Zhan, et al., 2018). In the presence of silicates several studies have shown a reduction in the nematode population of the root-knot nematodes which is agreed with our results. Lower number of eggs, juveniles, and galls (Roldi et al., 2017). As well as when silicon is made available galling (root-knot) caused by Meloidogyne spp. are less severe which resulting in less disease severity and slower disease progress. Fortunato et al. (2015).

Effect of Silicon attributed to its deposition in plant cell walls acts as a physical barrier to avoid the penetration of plant pathogens (Debona et al., 2017).

Many researchers result agreed with our results, applied Ascorbic acid in different way and their result also showed effect of AA on root-knot nematodes, application of ascorbic acid at 2000 ppm resulted in 100 % mortality of M. javanica juveniles after 7 days exposure and reduced the egg hatching Osman (1993).

In-vitro use of ascorbic acid 1250, 2500 and 5000 ppm increased the mortality of M. javanica juveniles up to 78.9 %. Infection criteria also decreased Abdel-Momen, et al. (2005) this results agreed with our result that AA effect on M. javanica result in a reduction in infection criteria.

Ascorbic acid also had practicable effect on decreasing infection criteria and increasing resistant, artificial increase in ascorbic acid concentration effected in transforms susceptible plants into more resistance against root-knot nematodes resistant ones (Farahat et al., 2015. Ascorbic acid have role in synthesis of mitochondrial hydroxyproline proteins, which control the development of cyanide-resistant respiration. Arrigoni, et al. (1979).

This result also cooperates with researches stated that SA drastically increased activity and synthesis of defense enzymes peroxidase and Phenol oxidase in tomato plants Infected by root-knot nematodes (Bakr & Hewedy, 2018). Results agreed with other researches their results showed that Salicylic acid increased the level of polyphenol oxidase and phenolic compounds, phenylalanine ammonia lays, peroxidase, accumulation in the root system of tomato after inoculation with M. javanica Mostafanezhad, et al. (2014) as well as SA application increased some defense compounds such as hydrogen peroxide and peroxidase activities in root of cucumbers infected by M. javanica. Siahpoush, et al. (2011). Salicylic acid SA promote and increase activity of many enzymes such as cytoplasmic peroxidase phenylalanine ammonia lyase and catalase, which they have effect in defense response of plants against infection. Nikoo et al. (2014).

Raise the number of phenolic substances by treatments in means more defenses and that is obvious in reducing infection criteria that is because phenolic compounds have role in defense against pathogens and according to reviews involve in resistant against nematodes attack Ohri and Pannu (2010). Ascorbic acid also had acceptable effect in increasing defense enzymes and phenolic substances this might because of the artificial increase in ascorbic acid concentration effected in transforms susceptible plants into more resistance against root-knot nematodes resistant ones. Ascorbic acid have role in synthesis of mitochondrial hydroxyproline proteins, which control the development of cyanide-resistant respiration. Arrigoni et al. (1979.)