Effect of Tomato yellow leaf curl virus (TYLCV) on the Content of some Mineral Elements in the Fruits of some Tomato (Solanum lycopersicom L.) Genotypes

This study was conducted to determine the reaction of 14 tomato (Solanum lycopersicom) genotypes to Tomato yellow leaf curl virus (TYLCV), and the effect of the virus on the fruit content of the genotypes Narcan, 123, and Summer 21. The field experiment was carried out in the western orchards of Samawah, Al-Muthanna Governorate, Iraq. All experiments analyzing mineral elements in tomato fruits were done in laboratories of Ghayat Al-Marefa Company, Babylon. The results of inoculation of the tomato genotypes with TYLCV and testing them by polymerase chain reaction (PCR) showed that all these genotypes were susceptible to the virus with the difference of those genotypes in the time of appearance and severity of disease symptoms. The genotypes Nahrayn and Summer 21 were the most severely infected with TYLCV (96 and 87%, respectively) compared with the other genotypes. It was also found that the virus had a clear effect on the decrease in the fruit genotypes contents of the samples tested (123, Narcan, and Summer 21) of mineral elements of iron and zinc that reached 26.80 and 28.88 mg/L, respectively, and substantially different from their given values, which were 32.94 and 34.73 mg/L, respectively, in unaffected plants. Moreover, results proved that the content of TYLCV-infected fruits decreased significantly (1.26 mmol. L-1) with a significant difference from its normal level in non-infected plants which was 1.8 mmol. L-1. Results also showed that TYLCV had a significant effect in reducing the concentrations of magnesium and phosphorous (0.244 and 0.248%), respectively, in the fruits of tomato genotypes compared with their concentrations in non-infected plants that were 0.348 and 0.383%, respectively.


Introduction
Tomato (Solanum lycopersicom L.) is one of the most important vegetable crop worldwide [1].Tomato fruits contain some vitamins such as A and C, and mineral elements such as iron and phosphorus.In addition, it is consumed fresh as well as used in many food industries [2].According to the report of the Food and Agriculture Organization, the production of tomato reached 186.9 million tons worldwide, and the cultivated area was 5.06 million hectares in 2020, and the developing countries in Asia contribute more than a third of the total production [3].In Iraq, the estimated tomato production for the year 2020 was about 754.8 thousand tons, an increase of 21.8% compared with the production 1259 (2023) 012093 IOP Publishing doi:10.1088/1755-1315/1259/1/012093 2 for the previous year, which was estimated at 619.5 thousand tons [3].As for the area cultivated with the tomato crop for the year 2020, it was about 128 thousand acres [4].In recent years, the spread and appearance of many diseases which specified to the production of this crop have increased due to the expansion of crop cultivation and the introduction of new technologies in agriculture that provided appropriate conditions for the spread of diseases such as fungal, bacterial and virus diseases such as Tomato yellow leaf curl virus (TYLCV) [5][6][7][8].TYLCV is one of the most common viruses in the world, including Iraq, inflicting severe economic losses of up to 100% in various commercial crops, including the tomato crop, which is considered the virus's primary host [9,10].Several methods were used to reduce the effect of the virus, including the use of resistant plant varieties and the treatment of plants with different extracts to increase plant tolerance to infection [11,12].This study aimed to investigate the effect of TYLCV on the contents of tomato fruits of some mineral elements in some tested genotypes.

Source of TYLCV Isolate
Tomato plants showing symptoms of viral infection represented by wrinkled, yellowing, and stunted leaves were collected from tomato cultivation areas in Al-Muthanna Governorate during the 2020-2021 agricultural season.Infected plants were planted in plastic pots (22 x 24 cm) and grown in the plastic house at the place of executing the experiment located in one of the orchards of Muthanna governorate after placing them in wooden cages (50 x 50 x 60 cm) each with a wooden base and its five sides covered with muslin.Young tomato plants (4-6 true leaves) were placed every 2-3 weeks with the infected plants in the same boxes for the purpose of transmitting the virus by whiteflies (Bemcia tabaci) to maintain the isolate as a source of the virus.

Virus Diagnosis using Polymerase Chain Reaction (PCR)
Total nucleic acid was extracted from tomato leaves using the kit (Cat No: FAPGK001, Favorgen, Taiwan) following the manufacturer's instructions.PCR amplification was performed using the kit (Go green master mix G2 Taq ® , Promega, USA) with a total volume of 25 µL containing 1 µL of each forward (GAATTCATG TCGAA GCGWCCA) and reverse (GAATTC TTAATTTK RTAYTGAA TCAGAA) primers [13] and one µL of nucleic acid (30) ng/ μL.PCR mixture was then adjusted to 25 μL with PCR grade water.The following conditions were used for PCR amplification: initial denaturation for 5 minutes at 94°C, followed by 35 cycles of final denaturation for 30 seconds at 94°C, annealing temperature for 30 seconds, initial extension 72°C for 1 minute, and final extension 72°C for 5 minutes at 52°C.PCRamplified products were separated by 1% agarose gel electrophoresis for 60 minutes at 120 V, 400 mA, visualized with Red safe staining under UV illumination and images were captured using Vilber Lourmat, Taiwan gel documentation system.

TYLCV Susceptibility Testing of some Tomato Genotypes
Fourteen tomato genotypes were tested for TYLCV resistance (Table 1).These genotypes' seeds were planted individually in cork dishes containing peat moss.twenty days of germination, the plants were separately transferred to plastic pots ( 8 × 31 cm) and grown in an insect-proof plastic.Plants (4-6 true leaves) were inoculated using with viruliferous whitefly insects (B.tabaci ).
Table 1.Tomato genotypes tested against TYLCV.Eighty-four hours after inoculation, the plants were sprayed with Acetamiprid 20% (Mospilan SL) and transferred to a plastic house with sealed doors made of muslin cloth to protect plants from insects.A control treatment was carried out by inoculating healthy plants of the same genotypes using nonviruliferous whitefly insects, and maintained in another plastic house under the same condition provided in the plastic house containing the plants inoculated with TYLCV.

Effect of TYLCV Infection on the Fruits Contents of some Mineral Elements
Digestion of tomato fruit samples and analysis of iron, zinc, calcium, magnesium, and phosphorous.The mineral content of plant fruits was measured by following the method of [14] for the purpose of digesting of tomato fruit samples.The fruit content of iron was measured according to the method described by [15].The presence of zinc in the tomato fruits was also determined using the kit (Cat.No. KA 1619) provided by Abnova company, Taiwan.However, the method described by [16] was followed to measure the calcium content of the fruits.The magnesium content of tomato was also measured using the method of [17].The phosphorus content of the fruits was measured by using the method of ammonium molybdate ([NH 4 ]2MoO 4 ) and ascorbic acid (C 6 H 8 O 6 ) by following the method described by [18].

Measurement of the Decrease Percentage in the Tomato Fruit Contents of Mineral Elements and Vitamins
The percentage decrease in the fruit content of mineral elements (iron, zinc, calcium, magnesium, and phosphorous) were determine using the following equation [19].

Statistical Analysis
All data obtained in this work were statistically analyzed by randomized complete block design (RCBD) using the Genstat statistical software version 18, and all means were compared at a probability level of 0.05.

Infection Severity
The results of assessing the reaction of several tomato genotypes to TYLCV revealed that all genotypes are susceptible to the virus, with differences in the length and severity of the subsequent pathological symptoms.It found that the highest infection severity was in the genotype Nahrayn which was not significantly different from the genotype 21 Summer, which showed an infection severity of 96% and 87%, respectively, while genotype 123 showed the least infection severity (40%), which significantly differed from all other genotypes.The other genotypes Super regina, Marina, Miami, Sata, Navya, Rochstar, Maysaloun, Madena, Nacran, Samar, and Bdoor showed infection severity ranging from 56-80%.The disease symptoms on the Summer 21 and Nahrayn genotypes appeared initially Following 15 days of viral inoculation, there was mild yellowing between the veins of the upper leaves and wrapping around the margins of the leaves, which spread to the lower leaves.These symptoms later developed into severe wrapping and wrinkling in the upper leaves with dwarfing and dropping of the plant flowers (Fig. 1).These results agreed with many researchers about the susceptibility of several tomato genotypes to TYLCV with differing of those genotypes to the severity of TYLCV infection [20,21] .Depending on the results of the infection severity obtained in the current study, the genotypes Narcan, 123, and Summer 21 were chosen based on the lower, medium and higher severity of infection.Three genotypes (123, Narcan, and Summer) were selected based on the lower, medium and higher severity of infection to determine the effect of TYLCV on the content of tomato fruits of the mineral elements zinc, iron, calcium, magnesium, and phosphorus.

Effect of TYLCV Infection on the Iron and Zinc Content of Tomato Fruit
Results demonstrated that the contents of the mineral elements of iron and zinc in the tomato genotypes were significantly decreased by TYLCV infection.Iron and zinc concentrations were 26.80 and 28.88 mg/ L, respectively, compared to healthy control, which were 32.94 and 34.73 mg/ L, respectively.The genotypes also varied in the fruit iron and zinc conrents.The genotype Summer 21 scored the lowest in zinc content among the other elements (24.89 and 26.89 mg/ L, respectively) compared to those recorded in non-infected plants (33.03 and 34.24 mg/ L, respectively).It was also observed that genotype 123 was the least affected in reducing the concentrations of both iron and zinc, with rates of 29.04 and 31.05mg/ L, respectively, which differed significantly from the content of non-infected plants (33.50 and 35.81 mg/ L, respectively (Table 2).The results also showed the difference in the percentages of decrease among the genotype content of the iron and zinc.the genotype Summer 21 scored the highest percentage of decrease (21.48 and 24.66.respectively), whereas the genotype 123 recorded the lowest percentage of decrease of iron and zinc that were 13.31 and 13. 27, respectively (Figure 4).

Calcium
The calcium content of tomato genotypes decreased significantly due to TYLCV infection at a rate of 1.26 mmol.L -1 , with a significant difference from its normal level with the non-infected plants that recorded 1.8 mmol.L -1 .there were also differences in the genotype content of calcium, it was found that the highest level of this element was in the genotype 123 (1.65 mmol.L -1 ); whereas the genotype Summer 21 was the lowest among the other genotypes (123 and Nacran) with an average of 1.44 mmol.L -1 (Table 3).The interaction between infected and non-infected genotypes revealed that the infected genotype summer 21 had the lowest content of calcium (1.1mmol.L -1 ) which differed significantly from the content of non-infected plants that reached 1.77 mmol.L -1 .Whereas, the highest level of calcium (1.45 mmol.L -1 ) was recorded in the genotype 123 which differed significantly from the content of non-infected plants (1.86 mmol.L -1 ).The results also showed the difference in percentage decrease between the three genotypes.Summer genotype 21 had the highest percentage of decrease that reached 37.78%, while the genotype 123 showed the lowest percentage of decrease ( 22.08%) (Figure 4).Table 3.Effect of TYLCV on the calcium content of fruits in some tomato genotypes.

Magnesium
As shown in Table 4, the results showed that TYLCV had a clear effect in reducing the concentration of magnesium (0.244%) with a significant difference from the non-infected plant, fruit content which was 0.384%.Genotype 123 recorded the highest levels of magnesium concentration (0.314%) compared with the 123 genotype that recorded the lowest average which reach 0.279%.However, it was noticed a significant decrease in the presence of magnesium in all tested genotypes infected with TYLCV compared with non-infected fruits and the lowest rate was in the genotype Summer 21 (0.244%) compared with the non-infected fruit content which was 0.342%.As for the genotype 123, it recorded the highest concentration in the infected plants (0.272%), with a significant difference from the non-infected plants, in which the magnesium rate was 0.356%.Moreover, the results showed a difference in percentage decrease among the three genotypes and the genotype Summer 21 recorded the highest rate of decrease which was 36.94%.Whereas, genotype 123 gave the lowest percentage of decrease that reach 23.69 5 (Figure 4).

Phosphorous
The results of analyzing the phosphorus content of tomato fruits showed that the concentration of phosphorus in all tested genotypes decreased significantly (248%) compared with non-infected fruits, which an average was 0.383%.It also found that genotype 123 among the tested genotypes recorded the highest average of 0.328% compared with genotype Summer 21, which recorded the lowest rate (0.299%) (Table 5).The results also showed that genotype 123 infected with the virus had the highest concentration of phosphorus, reaching 0.265%, compared to the non-infected plants which was 0.391%.Whereas, the Summer 21 genotype had the lowest concentration in infected plants (0.227%), compared with non-infected plants which reach 0.371%.The results also indicated a clear difference in the percentage of decrease among the three genotypes.The Summer 21 genotype showed the highest percentage of decrease (38.72%), while genotype 123 recorded the lowest percentage of decrease with an average of 32.2% (Figure 4).
Table 5.Effect of TYLCV on the phosphorus content of fruits in some tomato genotypes.It was clear from the results obtained in this study that infection of tomato plants with TYLCV has a role in a significant decrease in the content of the tomato plant fruits of the mineral elements iron, zinc, calcium, magnesium, and phosphorus with a significant difference from their normal levels that observed in the non-infected tomato fruits.[22,23] reported that viral infection has a clear effect on the plant content of some mineral elements, which negatively affect the performance of the physiological processes of the plant and appear in the form of deterioration and general weakness in plant growth.[24] showed that the content of tomato plants of the mineral elements calcium and magnesium decreased when the plants were infected with the Tomato mosaic virus (ToMV) with a significant difference from the content of non-infected plants.[25] proved that there was a high decrease in Telfairia contents of zinc, iron, calcium, magnesium, and phosphorus in the plants infected with Telfairia mosaic virus (TeMV) in comparison with non-infected plants.It also found that infection with TYLCV led to a significant decrease in the levels of calcium and magnesium in the leaves of tomato plants [8,21,23].

Figure 1 .
Figure 1.Pathological symptoms resulting from TYLCV infection in the tomato genotypes 123 and summer 21 tested in this study.

Figure 3 .
Figure 3.The percentage of infection severity in tomato genotypes examined against TYLCV in this study.

Figure 4 .
Figure 4.The relationship between the severity of TYLCV infection and the percentage decrease in zinc, iron, calcium, magnesium, and phosphorus in the tomato genotypes 123, Nacran, and Summer 21 tested against TYLCV.

Table 2 .
Effect of TYLCV iron and zinc contents in tomato fruits of three tomato genotypes.

Table 4 .
Effect of TYLCV on the magnesium content of fruits in some tomato genotypes.