Introduction
Tomato (Solanum lycopersicum L.) is one of the most widely cultivated vegetable crops worldwide. In terms of nutritional composition, tomato fruits contain 3% carbohydrates, 1.2% protein, 1% total fats, minerals (calcium, magnesium, phosphorous, potassium, sodium, zinc and manganese.), and different contents of vitamins (vitamins A and C, thiamine, riboflavin, niacin, pantothenic acid and pyridoxine) (Perveen et al. 2015; Melfi et al. 2018). Most cultivated tomato cultivars are exposed to infection with soil-borne pathogens, the most important of which is the fungus Rhizoctonia solani, one of the fungal pathogens transmitted through the soil and affects a wide range of plant families (Al-Hammouri et al. 2013). This pathogen also causes diseases in other members of family Solanaceae, especially in potato (Rafiq et al. 2020, 2021).
Effective control of this pathogen is difficult owing to the diversity of its host range, persistence of sclerotia formation in soil, lack of genetic resistance and limited efficacy of chemical fungicides (Zachow et al. 2011). Sumalatha et al. (2018) explained that some infected plants show symptoms of sunken watery spots that later turn into irregular brown spots on stems with the appearance of local necrosis on the bark. Mayo-Prieto et al. (2020) observed that the mycelium of this pathogen penetrates wound areas, leading to the rupture of the outer layer of the host’s epidermis.Tomato production faces enormous problems worldwide including lack of water resources, soil salinity and other abiotic stresses (Fahad et al. 2017; Zhou et al. 2019). These stresses have a detrimental effect on plant growth and development as they interfere plant morphological, physiological, biochemical and molecular responses (Rai et al. 2013; Abass 2016). Salt stress affects all major processes of agricultural crops such as germination, growth, photosynthesis, respiration, water content, nutrient imbalance, oxidative stress and yield (Yasin et al. 2018). Arif et al. (2020) reported that salinity enhances the content of reactive oxygen species in plant cells as a consequence of ion toxicity and ionic imbalance, which results in osmotic and ionic stress that disrupts the balance of nutrient absorption and damages the membranes and various internal structures. Fungal activity is affected by salt stress (Rilling 2004). Asghari et al. (2008) confirmed that salinity restricts the growth of mycelium through the harmful effect of salts. Similarly, Peat and Fitter (1993) indicated that salinity decreased the number of spores and availability of carbohydrates necessary for fungal growth. Likewise, Juniper and Abbott (2006) found that inhibits spore germination and mycelium growth. Salih and Al-Maarich (2016) reported that the pathogenic R. solani isolates RS1 and RS2 can grow under saline conditions with concentrations ranging from 6–16 dS m-1. The present study was conducted to investigate the biochemical response patterns of three tomato plant cultivars, namely, Salimah, Yassamen and Bushra to the interaction between the fungal pathogen R. solani and three NaCl levels under laboratory and greenhouse conditions. Materials and Methods Plant materials
The seeds used in this study were
tomato seeds of three varieties (Salima, Yassamen and Bushra) obtained from local markets and
predominantly cultivated in Zubair fields.
Salinity treatments
Three levels of sodium chloride
salt were used during field and laboratory experiments (2, 6 and 12 dS m-1). Distilled water was used to prepare the
irrigation water with the desired NaCl salinity based on an initial survey.
Isolation and Identification of R.
solani
The plant parts were collected from the affected areas of some tomato fields and nurseries planted in Zubair and Safwan districts, which showed symptoms of seed rot and seedling death, represented by rotting of seeds and stems of seedlings with a brown discoloration of the roots from light to dark, as well as wilting and yellowing of the leaves, especially the lower ones. The isolation on PDA medium containing Chloramphenicol (250 mg L-1) was done (Mohammed-Ameen et al. 2021); all inoculated plates were incubated at 25 ± 2ºC. The identification was confirmed depending on the characteristics of the fungal colony, the nature of branching of the new mycelium, the structures that it forms, the ability to form sclerotia, the formation of barrel cells and the presence of double-hole septa, using the taxonomic key of Parmeter and Whitney (1970).
Fungal nuclear staining All fungal isolates were stained according to Runion and Kelley (1993) using aniline blue and lactophenol.Pathogenicity test The fungal inoculum was prepared using millet seeds according Smiley et al. (2005); the pathogenicity trails were done on the seeds of Salimah tomato variety in petri dishes and pots experiment according to Bolkan and Butler (1974). For petri dishes trail, after seven days of incubation at 25 ± 2ºC the seed germination percentage was calculated; and for pots trails both seed germination and seedling damping off were measured; as well as; plant height, fresh and dry weight of shoot and root system after 45 days of inoculation.
The effect of salinity levels on fungal growth
The PDA medium was prepared using
sterile distilled water containing saline levels of 0, 2, 6 and 12 dS m-1, with the antibiotic Chloramphenicol at a
concentration of 250 mg L-1 and then sterilized with an Autoclave,
after the sterilization period, poured into sterile Petri dishes and inoculate
with a 0.5 cm diameter disc of PDA medium of each R. solani
isolate and incubated at a temperature of 25 ± 2ºC for three days. The radial
growth was measured every 24 h by taking the average of two perpendicular
diameters passing through the center of the disc and
until the growth in the control treatment reached the edge of the plate. The
percentage of radial toxicity was calculated according to the following
equation (Abass 2017):
%200707-0716_files/image002.png){kind=small}
Which C: fungal growth in control;
T: fungal growth in treatment. Additionally, the fungal dry growth inhibition undergoes
the effect of salinity was done according to Muhsin (1990) using PD broth.
Tomato varieties responses to R.
solani under salinity stress
The response of three varieties of
tomato was tested, namely Salima, Yassamen
and Bushra. In this experiment, sandy soil from one of the tomato farms in
Zubair was used and washed well to remove excess salinity and dried with peat
moss at a ratio of 1:3 then the soil was sterilized with an Autoclave for One
hour twice on two consecutive days. The inoculum of the pathogenic fungus RS3
loaded on local millet seeds was added at a rate of 1% (w/w) to the sterilized
soil, and it was planted in plastic pots (25 cm diameter), one pot contained 2
kg of sterile soil, and three days after adding the fungal inoculum to the soil
mixture. Three tomato cultivars were sown at a rate of 20 seeds pot-1
sterilized with 10% sodium hypochlorite solution for 2–3 min. As for the
control treatment, it was cultivated with the same steps without any addition;
and irrigated the pots with the saline levels used in the study 2, 6 and 12 dS m-1 and after two weeks of planting, the
percentages of germination and tomato seedlings damping off were calculated and
the infection rate was calculated. The interaction effect of pathogenic fungi
and salinity levels was studied on some indicators of plant growth, such as
plant height and fresh and dry weight for each of the shoot and root systems
after 45 days of planting. The experiment lasted for 60 days and at the end of
the experiment, some biochemical indicators were measured, including.
Photosynthetic pigments
The pigments chlorophyll a,
chlorophyll b and total chlorophyll were estimated and extracted based on the
method of Arnon (1949) and the content of carotenoids
and anthocyanins by Asare-Boamah et al. (1986)
and expressed in the unit (mg g-1).
Proline content
Proline content in leaf tissues was
measured by reaction with ninhydrin chromatically at 520 nm (Bates et al.
1973).
Hydrogen peroxide content
H2O2 levels
were measured in control and stressed laves tissues according to the procedure
of Sergiev et al. (1997). The hydrogen
peroxide content was calculated using the standard hydrogen peroxide curve.
Malondialdehyde content
MDA was used as a marker for membrane
lipid oxidation. MDA was extracted at 5% (w/v) with trichloroacetic acid (TCA),
absorbance was measured at 532 and 600 nm, and the MDA concentration was
calculated using the Extinction Coefficient (Heath and Packer 1968).
Total soluble carbohydrates
The method described in Watanabe et
al. (2000) was followed to estimate the carbohydrate content in leaf
tissues by interacting with the anthron reagent and
measuring the absorbance at a wavelength of 620 nm and the carbohydrates were
estimated using the standard glucose curve.
Statistical analysis
With three salt levels (2, 6 and 12
dS m-1 NaCl) and three tomato varieties (Salimah, Yassamen and Bushra),
the experiments utilized a completely randomized factorial design. All of the
tests were triplicates, and the data was analyzed
using SPSS-22 software for two-way analysis of variance (SPSS Inc., Chicago,
IL., USA). To examine significant variations between means, the least
significant difference (LSD) was employed. A P value of less than 0.05 was used
to determine statistical significance.
Results
Isolation and identification of R.
solani
Ten different isolates of R. solani (herein designated as RS1-RS10) were isolated
from different fields in Safwan and Zubair, Iraq.
Morphological and microscopic
characteristics of R. solani isolates
The 10 R. solani
incubated for 2 weeks on PDA culture medium in the dark at 25 ± 2°C. Examination of their
phenotypic characteristics revealed that they differ in appearance, consistent
with the findings of Yadav and Tiwari (2005), Lal and
Kandhari (2009) and Misawa and Kuninaga (2010) These
aforementioned studies reported that R. solani colonies differ in terms of growth, morphology
and colours, as well as in terms of their density and spread on the surface of
the culture medium. According to their microscopic features, the 10 R. solani isolates were found to have a different ability
to form swollen barrel-shaped cells called monilioid cells
or different manner of the hyphae branching (Fig. 1).
Nuclear staining of R. solani isolates
Microscopic examination showed that
the number of nuclei in the newly emerged hyphae cells of eight isolates,
namely, RS1, RS2, RS3, RS4, RS6, RS8, RS9 and RS10 was more than two nuclei per
fungal cell; the number of nuclei was between 3 and 9 nuclei/fungal cell (Table
1 and Fig. 2). By comparison the average number of nuclei in RS5 and RS7 was 2
nuclei/fungal cell (Fig. 2). Moreover, these two isolates were not pathogenic
to the tomato plants.
Pathogenicity trails
Results of pathogenicity trails on
Petri dishes (Table 2) showed that most of
the R. solani isolates examined herein
remarkably reduced the percentage of germination of tomato seeds on WA medium
by 16.6–66.6% compared with the control treatment, which reached 100%
germination rate (Table 2). The exception was the RS5 isolate (90.0%
germination rate), which was not significant differences (P < 0.05) from
that of the control treatment.
Pot experiments obtained similar
results. The difference in germination rates between the isolates and the
control treatment was not statistically significant. The control treatment
achieved 86.6% germination rate. By comparison the RS4, RS2 and RS3 isolates
had the lowest germination rate (40.0, 33.3 and 20.0%, respectively). The RS3
isolate showed the highest reduction in seed germination (80.0%) and seedling
damping off (14.90%). The RS2 isolate had 66.66% seed germination and 9.95%
seedling damping off. The RS4 isolate; the values were %200707-0716_files/image004.png)
Fig. 1: Colour and shape of colonies Rhizoctonia
solani isolates from soil and roots of tomato
plants on PDA culture media in the incubator at 25°C for one week
*The letters RS stand for Rhizoctonia solani and the number beside them represents the
isolate number
%200707-0716_files/image006.png)
Fig. 2: Nuclei numbers in hyphae of Rhizoctonia solani
isolates (X40)
* The letters RS stand for Rhizoctonia solani
and the number beside them represents the isolate number
60.0 and 7.19% for seed
germination and seedling damping off, respectively. In the control treatment, the
values were 14.0 and 0.00%, respectively. Therefore, the isolates whose rates
of seed germination and seedling damping off were not substantially different
from those of the control treatment were not (Table 3). Thus, RS3 isolate was
superior over the other isolates. Moreover, some of the growth parameters
including plant height and the fresh and dry weight of shoot and root systems
(Table 3) were considerably reduced. Clearly RS3 was the most pathogenic among
the 10 isolates, whereas the RS5 isolate was not pathogenic.
%200707-0716_files/image007.png)
Effects of salinity
levels on the growth of different R. solani isolates
in vitro
%200707-0716_files/image008.png)
The growth rates of the isolates
were slightly affected by the increases in salinity levels (Fig. 3). The
highest growth rate of 8.95 cm was recorded when salinity level was 12 dS m-1. This rate was not significantly different
from that of the control treatment (8.98 cm). When the salinity levels were 6
and 2 dS m-1 the radial growth rates of
the isolates was inhibited (8.62 and 7.78 cm), respectively, (Table 4).
Responses of different tomato
varieties to R. solani isolates and salinity
stress
Table 3: Pathological testing of isolates
of the fungus R. solani in tomato seed
germination (%) Seed decay (%), seedling damping off (%), plant height (cm) and
fresh and dry weight of the shoot and root system (mg) in plastic pots
|
Isolate
No. |
Seed
germination (%) |
Seed
decay (%) |
Seedling
damping off (%) |
Plant
height (cm) |
Fresh
weight (mg) |
Dry
weight (mg) |
||
|
Shoot |
Root |
Shoot |
Root |
|||||
|
Control |
86.66 |
14.00 |
0 |
15.16 |
548 |
49.66 |
38.33 |
6.50 |
|
RS1 |
46.66 |
53.66 |
3.40 |
13.30 |
459 |
38.66 |
29.33 |
2.66 |
|
RS2 |
33.33 |
66.66 |
9.95 |
10.41 |
400 |
36.33 |
28.66 |
4.50 |
|
RS3 |
20.00 |
80.00 |
14.90 |
8.90 |
301 |
12.00 |
14.33 |
2.33 |
|
RS4 |
40.00 |
60.00 |
7.19 |
11.33 |
483 |
38.00 |
32.33 |
4.70 |
|
RS5 |
86.66 |
13.66 |
0 |
14.50 |
540 |
45.00 |
35.00 |
5.40 |
|
RS6 |
80.00 |
20.00 |
0 |
11.16 |
433 |
30.50 |
15.20 |
3.20 |
|
RS7 |
60.00 |
40.00 |
0 |
13.16 |
523 |
20.00 |
36.33 |
4.20 |
|
RS8 |
73.33 |
27.00 |
0 |
10.50 |
477 |
33.80 |
29.86 |
3.10 |
|
RS9 |
73.33 |
27.00 |
0 |
11.40 |
343 |
35.60 |
24.00 |
5.00 |
|
RS10 |
53.33 |
46.66 |
0 |
13.70 |
413 |
34.20 |
26.43 |
4.30 |
|
LSD(P < 0.05) |
42.51 |
42.06 |
0.83 |
1.99 |
50.60 |
13.41 |
4.02 |
1.62 |
*The letters RS stand for Rhizoctonia solani and the number beside them represents the
isolate number
Table 4: Effect of salinity levels (dS m-1) on the radial growth rate (cm) of Rhizoctonia
solani isolates
|
Salinity level (dS m-1) |
Rhizoctonia solani isolates |
Average of salinity level |
||||
|
RS1 |
RS2 |
RS3 |
RS4 |
RS5 |
||
|
Control |
9.00 |
9.00 |
8.90 |
9.00 |
9.00 |
8.98a |
|
2 dS m-1 |
8.80 |
8.90 |
6.80 |
8.90 |
5.50 |
7.78c |
|
6 dS m-1 |
8.80 |
8.95 |
8.30 |
8.95 |
8.10 |
8.62b |
|
12 dS m-1 |
8.95 |
8.96 |
8.95 |
9.00 |
8.90 |
8.95a |
|
Average of isolates |
8.88a |
8.95a |
8.23b |
8.96a |
7.87c |
|
* The letters RS stand for Rhizoctonia solani
and the number beside them represents the isolate number
The isolates examined herein and
the salinity levels of irrigation water had a significant effect (P <
0.05) on the percentages of seed
germination and seedling death of the tomato varieties Salimah,
Yassamen and Bushra (Table 4). The Bushra cultivar
was more tolerant to salinity and fungal pathogen than the two other varieties.
Its average germination rate was 70.43%, which was significantly different from
that of Yassamen (61.50%) and Salima
(60.75%). Notably, the germination rate decreased from 94.08% in the control
treatment (i.e., without salinity and pathogen infection) to 83.16% when
the tomato plants were treated with the isolates and subjected to the salinity
level of 2 dS m-1. Moreover, the
germination rate further decreased to 52.16 and 27.50% when the tomato plants
treated with the isolates and subjected to salinity levels.
%200707-0716_files/image010.jpg)
Fig. 3: Effect of salinity levels (dS m-1) on the radial growth of Rhizoctonia solani isolates
* The letters RS stand for Rhizoctonia solani
and the number beside them represents the isolate number
he interaction between
the fungal isolates and the salinity levels was significantly decreased the
seed germination percentage of the tomato varieties. Salimah
was the most sensitive to the fungal isolate when the salinity level was 12 dS m-1. The germination decreased as the
salinity levels of the irrigation water increased, especially in the soil
contaminated with the fungus R. solani (Table
5).
The pathogenic effect of the R. solani isolates increased as the salinity levels
increased. The disease incidence in the control treatment was 5.36%. When the
tomato plants were treated with the R. solani isolates
and Table 5: Effect of the pathogenic fungus Rhizoctonia
solani and different salinity levels on
germination indicators and phenotypic characteristics of tomato varieties
|
Variety |
Treatment |
Seed
germination (%) |
Seedling damping off (%) |
Injury rate (%) |
plant height (cm) |
Fresh weight (mg) |
Dry weight (mg) |
||
|
Shoot |
Root |
Shoot |
Root |
||||||
|
Salimah |
Control |
88.50 |
8.44 |
5.72 |
9.44 |
244.75 |
42.25 |
89.00 |
5.12 |
|
2 dS m-1 + RS |
85.75 |
14.64 |
14.46 |
8.94 |
181.25 |
36.00 |
80.50 |
4.25 |
|
|
6 dS m-1 + RS |
48.75 |
35.83 |
30.83 |
8.21 |
125.00 |
28.00 |
34.00 |
1.75 |
|
|
12 dS m-1 + RS |
20.00 |
67.92 |
82.71 |
5.78 |
69.00 |
20.25 |
18.75 |
1.17 |
|
|
Yassamen |
Control |
95.25 |
5.23 |
5.20 |
7.75 |
183.25 |
27.50 |
30.50 |
3.37 |
|
2 dS m-1 + RS |
70.25 |
14.26 |
7.13 |
6.51 |
154.75 |
27.25 |
15.25 |
2.62 |
|
|
6 dS m-1 + RS |
52.25 |
52.25 |
26.70 |
6.36 |
81.25 |
15.00 |
11.37 |
1.50 |
|
|
12 dS m-1 + RS |
28.25 |
66.86 |
53.16 |
4.65 |
30.00 |
8.50 |
4.12 |
1.00 |
|
|
Bushra |
Control |
98.50 |
5.07 |
5.18 |
9.75 |
193.75 |
31.50 |
48.25 |
3.75 |
|
2 dS m-1 + RS |
93.50 |
10.71 |
5.41 |
7.63 |
158.25 |
21.50 |
40.50 |
3.25 |
|
|
6 dS m-1 + RS |
55.50 |
33.62 |
18.05 |
7.28 |
143.00 |
17.25 |
20.25 |
2.25 |
|
|
12 dS m-1 + RS |
34.25 |
47.23 |
38.20 |
6.90 |
111.25 |
13.00 |
9.93 |
1.75 |
|
|
LSD (P < 0.05) |
4.31 |
3.57 |
2.95 |
0.58 |
27.48 |
5.23 |
5.86 |
0.68 |
|
|
Average of varieties |
Salimah |
60.75b |
31.70b |
33.42a |
8.09a |
155.0a |
31.62a |
55.56a |
3.07a |
|
Yassamen |
61.50b |
34.65a |
23.04b |
6.31b |
112.3b |
19.56b |
15.31c |
2.12b |
|
|
Bushra |
70.43a |
24.15c |
16.70c |
7.88a |
151.5a |
20.81b |
29.73b |
2.75a |
|
|
LSD (P < 0.05) |
3.04 |
2.52 |
2.08 |
0.41 |
19.43 |
3.70 |
2.14 |
0.48 |
|
|
Average of treatments |
Control |
94.08a |
6.24d |
5.36d |
8.97a |
207.25a |
33.70a |
55.91a |
4.08a |
|
2 dS m-1 + RS |
83.16b |
13.20c |
8.99c |
7.69b |
164.75b |
28.25b |
45.41b |
3.37b |
|
|
6 dS m-1 + RS |
52.16c |
40.56b |
25.19b |
7.28b |
116.41c |
20.08c |
21.87c |
1.83c |
|
|
12 dS m-1 + RS |
27.50d |
60.66a |
58.02a |
5.77c |
70.08d |
13.91d |
10.93d |
1.30c |
|
|
LSD (P < 0.05) |
3.52 |
2.92 |
2.41 |
0.47 |
22.44 |
4.27 |
4.78 |
0.55 |
|
*The letters
RS stand for Rhizoctonia solani and the number
beside them represents the isolate number
Table 6: Effect of the pathogenic fungus
Rhizoctonia solani and different salinity
levels of on the biochemical characteristics of tomato varieties
|
Varieties |
Treatments |
Chl a (mg g-1) |
Chl b (mg g-1) |
Total Chl (mg g-1) |
Carotenoids (mg g-1) |
Anthocyanin (mg g-1) |
Carbohydrates (mg g-1) |
Proline (μg g-1) FW) |
H2O2 (μM. g-1 FW) |
MDA (nmole g-1) |
|
Salimah |
Control |
*2.89 |
0.84 |
3.74 |
1.40 |
0.060 |
2.00 |
1.23 |
1.195 |
0.077 |
|
2 dS m-1 + RS |
2.70 |
0.79 |
3.49 |
1.43 |
0.065 |
0.52 |
1.22 |
1.263 |
0.146 |
|
|
6 dS m-1 + RS |
2.33 |
0.79 |
3.12 |
1.57 |
0.074 |
0.52 |
1.31 |
1.570 |
0.195 |
|
|
12 dS m-1 + RS |
2.10 |
0.78 |
2.88 |
1.58 |
0.083 |
1.03 |
1.46 |
1.578 |
0.547 |
|
|
Yassamen |
Control |
3.12 |
1.02 |
4.14 |
1.72 |
0.054 |
1.52 |
1.14 |
0.016 |
0.042 |
|
2 dS m-1 + RS |
3.17 |
0.99 |
4.16 |
1.77 |
0.055 |
2.02 |
1.16 |
0.614 |
0.167 |
|
|
6 dS m-1 + RS |
2.83 |
0.93 |
3.76 |
1.80 |
0.062 |
1.53 |
1.25 |
1.179 |
0.235 |
|
|
12 dS m-1 + RS |
2.66 |
0.93 |
3.59 |
1.83 |
0.078 |
1.53 |
1.35 |
1.310 |
0.417 |
|
|
Bushra |
Control |
3.55 |
1.04 |
4.60 |
2.00 |
0.091 |
2.03 |
1.23 |
0.136 |
0.044 |
|
2 dS m-1 + RS |
3.46 |
1.02 |
4.49 |
2.08 |
0.095 |
2.02 |
1.25 |
0.173 |
0.057 |
|
|
6 dS m-1 + RS |
3.25 |
1.02 |
4.27 |
2.09 |
0.109 |
1.52 |
1.31 |
0.263 |
0.072 |
|
|
12 dS m-1 + RS |
3.08 |
0.92 |
4.00 |
2.11 |
0.162 |
2.03 |
1.43 |
0.628 |
0.126 |
|
|
LSD (P < 0.05) |
NS |
NS |
NS |
0.07 |
0.004 |
NS |
NS |
0.006 |
0.005 |
|
|
Average of variety |
Salimah |
2.50c |
0.80b |
3.31a |
1.49c |
0.071b |
1.02c |
1.30a |
1.402a |
0.241a |
|
Yassamen |
2.94b |
0.97a |
3.91b |
1.78b |
0.063c |
1.65b |
1.23b |
0.780b |
0.216b |
|
|
Bushra |
3.33a |
1.00a |
4.34c |
2.07a |
0.115a |
1.90a |
1.30a |
0.300c |
0.075c |
|
|
LSD (P < 0.05) |
0.08 |
0.03 |
0.09 |
0.05 |
0.003 |
0.24 |
0.01 |
0.004 |
0.004 |
|
|
Average of treatments |
Control |
3.19a |
0.97a |
4.16a |
1.75 |
0.070c |
1.85 |
1.20c |
0.449d |
0.055d |
|
2 dS m-1 + RS |
3.11a |
0.93b |
4.05b |
1.76 |
0.070c |
1.52 |
1.21c |
0.684c |
0.123c |
|
|
6 dS m-1 + RS |
2.80b |
0.91bc |
3.72c |
1.81 |
0.082b |
1.19 |
1.29b |
1.007b |
0.168b |
|
|
12 dS m-1 + RS |
2.61c |
0.88c |
3.49d |
1.80 |
0.108a |
1.53 |
1.41a |
1.170a |
0.364a |
|
|
LSD (P < 0.05) |
0.09 |
0.03 |
0.10 |
NS |
0.003 |
NS |
0.01 |
0.005 |
0.004 |
|
* The letters RS stand for Rhizoctonia solani
and the number beside them represents the isolate number
when the salinity level was 2 dS m-1 the disease incidence was 8.99. When the
salinity levels were 6 and 12 dS m-1, the
disease incidence was 25.19 and 58.02%, respectively.
Furthermore, the R. solani isolates, increasing salinity levels and their
interactions remarkably decreased the plant height and the fresh and dry
weights of shoot and root systems (Table 5).
Biochemical analyses of
the tomato varieties, responses to pathogen infection, increasing salinity levels and
their interactions revealed that the contents of chlorophyll a, chlorophyll b
and total chlorophyll significantly decreased. As the salinity level
further increased from 2 dS m−1 to
as 12 dS m−1, the contents of these
pigments further decreased (Table 6). However, the opposite
trend was observed in the contents of carotenoids and anthocyanins (Table 6).
As a response to
pathogen attack, salinity treatment and their interactions, the contents of
carbohydrates and proline accumulated at high levels. Proline
accumulation is one of the most important mechanisms that plants resort to
under the influence of salt stress. The concentration of proline substantially
increased with the increase in salinity levels.
Similarly, the contents
of hydrogen peroxide (H2O2) and malondialdehyde (MDA) in
the tomato varieties significantly increased in response to pathogen attack,
salinity treatment and their interactions. The content of H2O2 in tomato leaves significantly as salinity levels increased.
Infection with the isolates at the salinity level of 12 dS
m-1 resulted in the highest H2O2 content of 1.170 µmol g-1. When the salinity
levels were 6 and 2 dS m-1, H2O2
content was 1.007 and 0.684 µmol g-1,
respectively.
In the tomato leaves,
the content of MDA, which is the product of the peroxidation of polyunsaturated
fatty acids in cell membrane, increased as salinity levels increased because of high level of oxidative stress. The average MDA content
substantially increased from 0.123 µmol g-1 to 0.364 µmol
g-1 as the salt level increased from 2 to 12 dS m-1 in the presence of the R. solani isolates. The interaction between salinity level
and the R. solani isolates had a significant
effect on the MDA content.
Discussion
R. solani
isolates
were isolated from different
parts of tomato plants collected from various areas in Basrah Province, Iraq. On average, the RS5 and RS7
isolates, had 2 nuclei per ungal cell. Thus, these
isolates were not pathogenic to the tomato plants. By comparison, other
isolates had multiple nuclei. Hence, they were pathogenic to the tomato plants.
These results were consistent with those found by Mirmajlessi
et al. (2012) and Mustafa et al. (2021) who reported that
multinucleate R. solani isolates are
pathogenic to plants. Several studies indicated that binucleate R. Solani isolates are non-pathogenic and thus could be
used in the biocontrol of pathogenic isolates (Elsharkawy
et al. 2014). Pathogenicity trails proved that most of the R. solani isolates examined herein had high pathogenicity
effects. The present work and previous studies
confirmed that fungal isolates have the ability to reduce the germination rate
of the plant seeds (Li et al. 2019).
The difference in the
pathogenicity among the isolates examined in this study could be attributed to
variation in the amount of toxic substances they secrete. Although these toxic
substances, are chemically similar they differ quantitatively. Wyllie (1962)
suggested that the difference in the pathogenicity of R. solani
isolates may be due to their different capabilities to
parasitize on the seeds directly. Highly pathogenic isolates cover seeds with
mycelium, thereby preventing them from germinating. The
protease enzyme plays a major role in determining the pathogenicity of R. solani (Ramezani 2008).
The superiority of the
RS3 isolate over the other isolates may be due to difference in the ability to
secrete degrading enzymes such as cellulase and pectinase. Moreover, it could
be attributed to the secretion of amylases enzymes that leads to cell killing
and turn the colour of seeds into dark brown (Ravjit et
al. 1999; Mahmoud et al. 2007). Furthermore, it could be attributed
to differences in their ability to secrete some phytotoxin compounds that can
kill seed embryo, such as phenyl acetic acid and its hydroxylated derivatives,
beta-hydroxy acetic acid and para-hyderoxyacetic acid
(Mandava et al. 1980).
The RS3 isolate was the
most pathogenic whereas the RS5 was not pathogenic. This seeming discrepancy
was also noted by other researchers. The effects of
different isolates of the R. solani on the
growth of tomato plants vary, some have negative effect, whereas others promote
plant growth (Macnish et al. 1995; Inoue et
al. 2002).
The results of the evaluation of the effects of salinity levels on fungal growth obtained in this study, were
consistent with the findings of Regragui and Lahlou (2005); Mustafa et al. (2021). The
interaction between the fungal isolates isolated herein and the salinity levels
was significant decreased the seed germination rate of the tomato varieties
examined herein; This result may be due to the fact that salinity
reduces and delays germination, a condition that increases the chance of fungus
attacking the seeds (Li et al. 2019).
Growth parameters,
including plant height and the fresh and dry weights of shoot and root systems,
significantly decreased. Kaya and Kirnak (2001) and Kutuk et al. (2005) indicated that increasing soil
salinity levels decrease the fresh and dry weights of the shoot and root
systems of tomato plants, because of the decrease in the ability of the plants
to absorb water and nutrients as the ions involved in the composition of salts leach
into plant tissues, thereby impeding water transfer (Yeo 1998).
The decrease in the
contents of chlorophyll a, chlorophyll b and total chlorophyll could be the
results of the generation chlorophyllase, which is responsible for chlorophyll
degradation. Furthermore, the decrease in contents of these pigments could be
that result of changes in the composition of the chloroplasts at high salinity
levels, thereby, that degraded plastid proteins and reduced chlorophyll
contents thereby inhibiting electron transport (Tuna et al. 2008).
The contents of
carotenoids and anthocyanins increased in response to pathogen attack and
salinity stress. Previous studies indicated that salinity increases carotenoids
content in tomatoes, because salt stress enhances carotenoids accumulation.
Krauss et al. (2006) showed that the reduced leaf area caused by growth
inhibition under salinity stress leads to increased carotenoid accumulation.
Anthocyanin pigments represent a subgroup of plant flavonoids and play
important roles in plants as photoprotective pigments in shoot tissues for ultraviolet
and high light absorption, and they also serve as antioxidants. Anthocyanins
accumulate in plant tissues in response to various types of abiotic stresses,
including osmotic stress, salinity, and high temperatures (Pourcel
et al. 2007; Mouradov and Spangenberg 2014).
Carbohydrates and
proline accumulated at high levels in response to pathogen attack, salinity
treatment and their interaction. Previous studies reported that high salinity
levels and fungal attack increase carbohydrates production and accumulation
(Sarwar and Ashraf 2003). The content carbohydrate increased in the tomato
leaves as the salinity level increased. Microorganisms adapt to salt stress by
accumulating organic compounds (proline, glycine and betaine) and inorganic
compounds soluble in cells, including potassium cations (Sagot
et al. 2010).
Proline accumulation is
one of the most important mechanisms that plants resort to under the influence
of salt stress. Its accumulation in plants under excessive salinity levels is a
primary response to maintain osmotic pressure in cells due to the decrease in
the activity of oxidative enzymes (Sudhakar 2001). Proline concentration
considerably increased as salinity levels increased. Proline plays the role of
an effective osmotic protector and is the key to protection against external
stresses and is also known as a salt tolerance limiter (Dogan et al.
2010).
The increase in salinity
levels resulted in higher MDA contents in the plant leaves. MDA is the product
of the peroxidation of polyunsaturated fatty acids in cell membrane, and its
content in plants increases under a high level of oxidative stress (Pan et
al. 2006). The interaction between salinity level and the R. solani isolates had a remarkable effect on the MDA
content. This result was consistent with that of Giannakoula
and Ilias (2013) who observed that lipid peroxidation
increased in tomato plants treated with 150 mM NaCl. As NaCl content
increased, MDA production was higher by twofold than that of the control
treatment. Moreover, H2O2 concentration linearly changed
with the increase in NaCl content. Kaushik and Roychoudhury
(2014), and Foyer (2018) indicated that the production of reactive oxygen
species in affected plants affects physiological aspects and growth by
increasing damage to membranes (lipid peroxidation), proteins, carbohydrates,
nucleic acids and plant pigments while decreasing seed viability and root
growth. In the manner, pathogens can destroy the defence system of
host plants and successfully establish.
Conclusion
Ten R. solani
isolates were isolated form symptomatic leaves and crown parts of tomato
plants. Pathogenicity test proved the virulence effect of R. solani isolates on the sensitive tomato variety Salimah. There salinity levels as 2; 6 and 12 dS m-1 of water salinity were selected to
examine their effect on tomato physiology and their responses to pathogen
attack. Results revealed the pathogenic effect of R. solani
alone or in interaction with salinity levels on tomato varieties on germination
and plant height, fresh and dry weight of shoots and roots. Additionally, a
decrease in the chlorophyll content with the increase of salt concentrations
was observed, and an increase in the carotenoids, anthocyanins, carbohydrates
and proline contents in the leaves. A significant correlation between the
salinity levels and H2O2 accumulation was revealed at the
interactions treatments between R. solani and
salinity levels.
Acknowledgements
None.
No funding to declare.
Author
Contributions
AAM
conducted the experiments and collected the samples, KMA interpreted the
results and statistically analyzed, MHA supervised research and provided
guidelines for writing manuscript and write the manuscript.
Conflicts
of Interest
All
authors declare no conflicts of interest.
Data
Availability
Data
presented in this study will be available on a fair request to the
corresponding author.
Ethics
Approval
Not applicable in this paper.
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