Penetration and Development of Meloidogyne javanica on Four Pistachio Rootstocks and Their Defense Responses
Article Category: Research Paper
Online veröffentlicht: 20. Jan. 2023
Seitenbereich: -
Eingereicht: 08. Juni 2022
DOI: https://doi.org/10.2478/jofnem-2022-0056
Schlüsselwörter
© 2022 Walter de Gruyter GmbH, Berlin/Boston
This work is licensed under the Creative Commons Attribution 4.0 International License.
Root-knot nematodes,
In Iran, two species, namely,
Given the high economic impact of parasitic nematodes, numerous strategies have been developed for nematode control in agriculture. However, the application of chemical nematicides results in intensive environmental hazards (Zhang et al., 2017). Another way of controlling root-knot nematodes is the use of biocontrol agents. The effect of some probiotic bacteria as biocontrol agents of
Several types of host–nematode relationships have been studied, such as carrot-
Madani et al. (2012) evaluated the egg mass and gall indices (EMI and GI) in Pistachio-
The objectives of the present study were to (i) evaluate penetration, development, and reproduction of
Several infested soil samples and roots having
Seeds of Badami, Ghazvini, and Sarakhs rootstocks (
The pistachio seedlings were inoculated with 3,000 J2 per plant (Pi = initial inoculum level) at the 8 to 10 leaf stage. The J2 were introduced into 3-cm-deep holes around the collar region of the plants. The inoculated pots were arranged in a complete randomized block design with six replications. They were irrigated and fertilized, as needed, and maintained under greenhouse conditions (25–30°C) with a 16-h light and 8-h dark regime. The pistachio seedlings were harvested at 120 dpi. The stem length and fresh root weight of pistachio seedlings, the nematode GI, EMI, and reproduction factor (RF) were measured. The roots were stained with Phloxine B and evaluated for GI or EMI, which had 1: 1 to 2 galls or egg masses; 2: 3 to 10 galls or egg masses; 3: 11 to 30 galls or egg masses; 4: 31 to 100 galls or egg masses; and 5: >100 galls or egg masses per root system (Hartman and Sasser, 1985). Eggs and juveniles were extracted from roots, as described by Hussey and Barker (1973), using 1% NaOCl solution and a blender, instead of manual agitation. The RF was estimated as RF = FP/IP, where FP is the final nematode population and IP is the initial nematode population (IP = 3,000 JV2s) (Oostenbrink, 1966).
Furthermore, the resistance index was measured according to Sasser et al. (1984) scale (Table 1).
Resistance rating scale for
| GI or EMI | RF | Resistance degree |
|---|---|---|
| 1 | <0.1 | Highly resistant |
| 2 | £1 | Resistant |
| 3 | >1 | Moderately resistant |
| 4 | >1 | Susceptible |
| 5 | >10 | Highly susceptible |
EMI, egg mass index; GI, gall index; RF, reproduction factor.
Ten seedlings of each pistachio rootstock were inoculated, as mentioned in resistance assays. This experiment was conducted with two replicates. J2 were introduced into 3-cm-deep holes around the collar region of the plant. Distilled water without nematodes was added to the control plants. Roots were removed from the pots and were carefully washed with tap water at 2 dpi, 4 dpi, 6 dpi, 10 dpi, 15 dpi, 21 dpi, 28 dpi, 35 dpi, and 45 dpi. The roots were stained with acid fuchsin to detect J2 penetration, their localization, and subsequent development within the roots. The roots were immersed in 3% NaOCl solution for 5 min, followed by rinsing with tap water to remove excess NaOCl (the root epidermis was dark, and for proper bleaching and preventing of damage to the cortex, the amount of NaOCl was increased, and the duration was reduced). The roots were then stained with 1 ml of 3.5% acid fuchsin stain (Byrd et al., 1983). The mixture was heated to the boiling level, then cooled at room temperature, and washed in running water. The roots were kept in acidified glycerin and were gently shaken for two days. After staining, root segments were observed under a stereomicroscope, and those parts showing nematode infection were mounted on a slide for observation under a light microscope (Olympus, BH-2).
Statistical analysis of the data obtained from different tests was performed using SPSS software (version 26), and the means were compared using Duncan’s multiple range test at a 95% confidence level.
The stem length of the four non-inoculated pistachio rootstocks was not the same, and, Badami, Ghazvini, Sarakhs, and Baneh ranked from longest to shortest, respectively. All the treated rootstocks had a significant stem length reduction at 120 dpi compared to their controls (Fig. 1A).
Figure 1
A- Effect of nematode infection on stem length of four pistachio rootstocks: Badami, Ghazvini, Sarakhs, and Baneh. B- Effect of nematode infection on fresh root weight of four pistachio rootstocks: Badami, Ghazvini, Sarakhs, and Baneh. The means with the same letter do not have significant differences according to Duncan’s multiple range test at the 95% confidence level.
The fresh root weight differed between the tested plants. The highest fresh root weight in non-inoculated was found in Badami (2.25 g) and Ghazvini (2.07 g) rootstocks. Baneh had the least root weight in healthy controls (0.92 g). At 120 dpi, the fresh root weights of Badami, Ghazvini, Sarakhs, and Baneh rootstocks were 1.32 g, 1.41 g, 0.99 g, and 0.69 g, respectively. The reaction of them was not the same after infection with
The root-knot nematode resistance was evaluated based on the GI, EMI, and RF indices. The results of corresponding statistical analysis are shown in Table 2. Badami exhibited high levels of gall and egg mass indices (GI or EMI = 4), and the nematode had a high level of reproduction (RF average = 4.43), so it is ranked as the susceptible rootstock to
| Pistachio | EMI (g root)-1 | GI (g root)-1 | RF | Resistance |
|---|---|---|---|---|
| rootstock | ||||
| Badami | 33.41a | 64.18a | 4.43a | S |
| Ghazvini | 12.26c | 29.98b | 1.37b | MR |
| Sarakhs | 16.49b | 27.14b | 1.27b | MR |
| Baneh | 8.49d | 17.23c | 0.25c | R |
EMI, egg mass index; GI, gall index; MR, moderately resistant; R, resistant; RF, reproduction factor; S, Susceptible. The means with the same letter do not have significant differences according to Duncan’s multiple range test at the 95% confidence level.
The results of acid fuchsin staining of the roots showed a high number of J2 were able to penetrate the root tip of all cultivars at 2 dpi (Figs. 2a,b). At 2 dpi and 4 dpi, the number of J2 that penetrated Badami rootstock was significantly higher than other rootstocks. At 4 dpi, Badami had the greatest penetration rate of the juveniles, but for Ghazvini, Sarakhs, and Baneh, the highest penetration rate was at 6 dpi. After 4 dpi, the number of penetrated J2 slightly decreased in Badami roots, but in Ghazvini, Sarakhs, and Baneh, it rapidly reduced after 6 dpi. Penetration rates of the J2 into Sarakhs and Baneh roots were lower than those in Badami and Ghazvini at 6 dpi. The least penetration rate was observed in Baneh (Fig. 3).
Figure 2
a,b: Penetration of J2 of
Figure 3
Dynamics of penetration and development of
After penetration, the J2 immediately entered the vascular cylinder from the longitudinal growth area of the root (Figs. 4a,b) or migrated in groups through the cortex to reach thicker parts of the root (Figs. 4c,d). The cortex cells adjacent to the J2 were slightly swollen, but the giant cells did not form. The giant cells were only formed in the vascular cylinder and formed around the nematode head region. The first giant cells were observed in Badami at 4 dpi; for the other three rootstocks, they were observed at 6 dpi. The macroscopic symptom of root swelling was observed from 10 dpi onward. The first “midstage” or swollen juveniles (terminology after Windham and Wilhams, 1994) were observed at 4 dpi, but with lower extent in Ghazvini, Sarakhs, and Baneh cultivars (Figs. 3,4e). The first females were seen in Badami at 21 dpi, in Ghazvini and Sarakhs at 35 dpi, and in Baneh at 45 dpi (Figs. 3,4f). The first J2 from the new generation were obtained in Badami at 28 dpi and in Ghazvini at 35 dpi, but there were no J2 of the new generation seen in Sarakhs and Baneh at 45 dpi. So, there was more delay in nematode development in Baneh, Sarakhs, and Ghazvini, respectively, compared to that in Badami.
Figure 4
Development of
An initial defense reaction was a hypersensitive response (HR)-like reaction observed in moderately resistant (Ghazvini and Sarakhs) and resistant (Baneh) rootstocks at 4 dpi and 6 dpi, respectively. It was characterized by necrosis of cells directly affected by nematode in the cortex. This HR-like reaction led to death of the J2 (Fig. 5a); however, some of the J2 died in the cortex without any HR response (Fig. 5b).
Figure 5
Resistance reaction of pistachio rootstocks roots to
Another early defense response observed in moderately resistant (Ghazvini and Sarakhs) and resistant (Baneh) rootstocks at 10 dpi and 6 dpi, was programmed cell death (HR), which occurred in the vascular cylinder during the formation of feeding sites. It prevents development of nematodes and feeding sites and has led to nematode death (Figs. 5c–e).
Moreover, this reaction was more prevalent in Baneh than Ghazvini and Sarakhs. HR was not observed in Badami rootstock.
The late defense reaction observed in all of the rootstocks was malformation and destruction of the giant cells occurred from 15 dpi onward. It was characterized by browning of feeding sites close to the nematode head, indicating necrosis of tissues. Also, no alive nematodes were found in these galls (Fig. 5f).
During the present study, the reaction of three rootstocks and one wild-type pistachio was assessed against the infection by
According to Roberts (2002), resistance of plants against nematodes refers to the ability of the plants to inhibit the development or reproduction of nematodes. The GI and EMI are considered good guides to evaluate resistance via the measurement of nematode establishment and reproduction in the host, respectively (Gomes et al., 2015). Soares et al. (2018) urged that the RF is a reliable factor in selecting resistant genotypes because it considers the final and initial populations of the nematode. The aforementioned indices are reliable standards for resistance assessments against root-knot nematode infection and have been previously applied in screening for
In the previous and only available relevant study by Madani et al. (2012) using pistachio–
Mechanisms of resistance to root-knot nematodes can be categorized into pre- and post-infection steps (Anwar and McKenry, 2002; Bendezu and Starr, 2003). The pre-infection resistance is defined by the failure of nematode to penetrate the root (Bendezu and Starr, 2003; Pegard et al., 2005). During the post-infection resistance, the penetrated juveniles fail to develop to females (Anwar and McKenry, 2002). During this study, the J2 of
After 4 dpi, the J2 number slightly decreased in Badami, but in Ghazvini, Sarakhs, and Baneh, it was rapidly declined after 6 dpi. Voisin et al. (1999) showed some of penetrated J2 into the resistant cultivars of myrobalan plum, emigrated from the root, and entered other host plants. The juvenile death in response to host defense responses or their emigration has already documented in the case of the resistant cultivars (Cabasan et al., 2012; Phan et al., 2018). The dead juveniles in the cortex were observed during the present study as well. Therefore, the more reduction of J2 in Baneh, Sarakhs, and Ghazvini than that in Badami rootstock showed there are resistance sources in them.
The first females were seen in Badami at 21 dpi, in Ghazvini and Sarakhs at 35 dpi, and in Baneh at 45 dpi. So, the nematode life cycle was delayed in Ghazvini and Sarakhs (relatively resistant) and Baneh (resistant). The delay in the life cycle of root-knot nematodes was observed in the resistant genotype of
One of post-infection mechanisms of resistance is HR, a type of programmed cell death that is induced after the invasion of avirulent pathogens to prevent the spread of biotrophic pathogens (Huysmans et al., 2017). It has been observed at three different phases of plant parasitic nematode (PPN) infection in resistant plants: (1) in the cortex and epidermis during penetration and migration, (2) in vascular tissues during the initiation of feeding cell formation, and (3) in cells adjacent to developing feeding cells (Sato et al., 2019). The first phase of HR (herein named HR-like reaction), which occurred in the cortex and vascular cylinder at 4 dpi and 6 dpi was observed in Ghazvini, Sarakhs, and Baneh (moderately resistant and resistant), but not in Badami (susceptible). This reaction, which prevents the migration of J2 in the root, has already been observed in resistant cultivars of coffee (Lima et al., 2015), pepper (Pegard et al., 2005), and peanut (Proite et al., 2008) against root-knot nematode infection. Pegard et al. (2005) characterized this HR-like reaction displaying blue autofluorescence under UV light, proving the accumulation of phenolic compounds. This early HR reaction (in the epidermis and root cortex) expedites the success of the plant to overcome the nematode invasion (Bleve-Zacheo et al., 1998; Castagnone-Sereno et al., 2001). Some of J2 were however able to escape the initial defense reaction. In such cases, the secondary HR reaction occurred in the vascular cylinder, where they tried to form initial feeding cites. The secondary HR aims to prevent the formation of giant cells. During this phase, necrosis occurred in cells opposite to the nematode head. This reaction was mostly observed in Ghazvini, Sarakhs, and Baneh and, to a less extent, in Badami, at 6 dpi to 10 dpi, and has been previously reported in tomato at 1 dpi (Williamson, 1999), in pepper at 1 dpi to 3 dpi (Pegard et al., 2005), in soybean at 2 dpi to 3 dpi (Kaplan et al., 1979), and in coffee at 4 dpi to 6 dpi (Anthony et al., 2005). The third and the last HR, which was the last defense reaction against
The expression of HR-like reaction and early and late HR defense responses in infected roots could indicate that multiple genes are likely to be involved in the resistance of pistachio rootstocks. The two latter HR reactions triggered in susceptible Badami rootstock as well, but in a slow rate and to a less extent, compared to those in moderately resistant and resistant rootstocks. However, none of studied pistachio rootstock were completely resistant to
During the present study, both host pistachio plants and root-knot nematode-related indices were assessed in greenhouse experiments, and as the result, the Badami cultivar was proven to be susceptible, two cultivars Ghazvini and Sarakhs were proven as relatively resistant, and the wild pistachio, Baneh, was proven to be resistant against