Impact of Nitrogen Fertilization Types on Leaf Miner, Liriomyza trifolii Infestation, Growth and Productivity of Pea Plants under Pest Control Program

Pea, Pisum sativum L. is one of the most important cool season legume crops belonging to the Leguminosae: Fabaceae in Egypt for local consumption and exportation (Elsharkawy, 2013). Pea pods and seeds  are considered  important source in human nutrition, being its pods and seeds contain a great amount of protein, carbohydrates, vitamins, phosphorus, iron, magnesium, calcium, and amino acids, minerals supplement fibers and antioxidant compounds (Urbano et al., 2003 and Mohan et al., 2013). Besides, pea plants considered as a soil fertility building crop through N₂ fixation in symbiotic association with beneficial bacteria (Alam et al., 2010 and Elsharkawy 2013). Economically, pea is predominately exported vegetable crop from Egypt to numerous countries over the world (Zaghloul et al., 2015 and Chaney, 1995). The total cultivated area of green and dry peas in Egypt was about 46134.02 acres in 2020 with total production 153,440 tons according to FAO (2020). The legume crops were infested by different leaf miners causing significantly damages in leaves. Thesevere pests of vegetable plants are Liriomyza species (Diptera: Agromyzidae) which infested exceeded than 20 crops belonging to Fabaceae, Cucurbitaceae, Solanaceae, Brassicaceae, Malvaceous, Amaryllidaceae and Amaranthaceae families (Foba et al., 2015 and Foba et al., 2016). Leaf miner flies of Liriomyza were injurious dipterous insects infesting vegetable and horticultural plants (Jones et al., 1986 and Parrella et al., 1987). Liriomyza species are the most economic importance pests as Liriomyza trifolii, L. pisi and L. congest (Burgio et al., 2005 and El-Sayed et al., 2007). It caused direct damages to plants and indirect by transmitting many microorganisms that are of economic importance to many crops. Feeding by adults, and the mining of leaves by larvae decreased photosynthetic tissue, dehydration and withering of leaves leading to significant losses in yield (Facknath and Lalljee, 2005).

The serpentine leaf miner, Liriomyza trifolii (Burgess) (Diptera: Agromyzidae) is a perennial pest of leguminous crops in the Mediterranean region especially in Egypt (Foba et al., 2015, Shaalan and El-Ghanam, 2016 and Bayoumy et al., 2018). Moreover, polyhouse crops also infested with L. trifolii (Vashisth et al., 2013). In addition, Doss et al. (1992) and Abdel-gawad (2008) reported that leaf miner is one of the most important field pests to vegetable and horticultural crops. Punctures or damages were made on leaves by L. trifolii larvae three to four days after oviposition and grow as the larvae matures (Capinera, 2001 and Wilkerson et al., 2005). The mine is irregular, can greatly depress the photosynthesis in plant, and also causes premature leaf drop (Bueno et al., 2007). Also, wounding in leaves by different stages of the pest allows to infect plants with bacterial and fungal diseases (Abou-fakhr Hammad and Nemer, 2000).

On the other side of the research, Nitrogen (N) is an essential macronutrient for plant development, chlorophyll, enzymes, and protein syntheses in plant, it is main fertilizer for plant development and yield of crops (Bhuvaneswari et al., 2014). Nevertheless, nitrogen is a major essential element for all organisms and a constituent of nucleic acids, proteins and other indispensable organic compounds (Ohyama, 2010). Nitrogen is an important for amino acid synthesis and proteins, which are determining nutrients for insect survival (Rashid et al., 2017). Nitrogen element could influence the presence of insects, positively or negatively. Nitrogen fertilizer increased the level of the population of piercing-sucking pests, including the leaf miner, L. trifolii (Elsayed et al., 2021). There are three types of chemical nitrogen fertilizers, most growers use it for enhancing growth and productivity of crops due to its easy and rapid availability to plants. The first is ammonium sulphate [(NH₄)₂ SO₄] (20.6% N and  24% S), it was applied by growers primarily where they need supplemental N and S to meet the nutritional requirement of growing plants and decrease pH of the soil (Nasim et al., 2012). The second is urea H₂NCONH₂ (46.5%N) where urea nitrogen enters the plant either directly, or in the form of ammonium or nitrate after urea degradation by soil microbes, and the third is ammonium nitrate NH₄NO₃ 33.5%  N (Chien et al., 2011 and El Mantawy, 2017).

Fertilization play important role in the plant physiology and growth characteristics, and it may be related to a higher incidence of a pea leaf miner (Nestel et al., 1994). Herbivorous insects frequently choose their plant hosts based on the availability of mineral nutrients since nutrients change the chemical composition, anatomy, morphology and phenology of plants (Han et al., 2014; Oliveira, 2014 and Uesugi, 2015). Availability of mineral nutrients usually influence host-plant selection by herbivorous insects as nutrients alter plants’ chemical composition, morphology, phenology and anatomy (Han et al., 2014; Oliveira, 2014 and Uesugi, 2015). High quantity of nitrogen in soil increased leaf content of amino acids and proteins, as well as improving of vegetative growth, delaying maturity and lignification of plant tissues, as well as making the plant was more attractive to herbivorous insects (Coqueret et al., 2017).

Rodrigues et al. (2009) found negative relation between nitrogen and population of leaf miner, Liriomyza sp. and its control by biological agents, Metarhizium anisopliae and Beauveria bassiana on bean, Phaseolus vulgaris influencing by N fertilization rates. Additionally, the highly percentage of leaf miner attack in common bean under N excessed was recorded by (Cruz et al., 2012). As well as, soil-applied complex fertilizer effect on the insect-host plant interaction between L. trifolii infestations and potato plant, where the increase of nitrogen concentrations in potato leaves increased the L. trifolii development (Facknath and Lalljee, 2005). The control of the leaf miner, Liriomyza sp. has been tackled on faba bean by El-Nahal and Assem,(1970) and Li et al. (2009) on pea crop. Chemical control was conducted by Cyromazine (insect growth regulator used as an insecticide) on controlling Liriomyza sp. in kidney beans (Xu et al., 2009), and neem extract on tomato (Salas and Mendoza, 2001). A direct relationship between synthetic NPK fertilizer and bean fly (agromyzid) densities was reported by Letourneau (1994). Moreover, the impact of N levels on epidemiology of pests in winter wheat cultivars grown under conventional, integrated, and biodynamic cropping systems in Netherlands with a high N levels stimulated epidemics of leaf miners as reported by (Daamen et al., 1989). From the previous view , the objective of this research is to determine the effect of nitrogen types on the infestations with the serpentine leaf miner, Liriomyza trifolii Burgess (Diptera: Agromyzidae), as well as , growth and  productivity of pod  yield of pea plants  cultivated  under the conditions of proposed control program.

MATERIALS AND METHODS

Field site description

In a field experiment conducted in a private farm in Kafer Singerg, Menoufia governorate, Egypt, during winter season on 26^th of September 2021 and 2022, respectively depending on the day by the year.  Pea (Pisum sativum L.) cv. Master B was grown in a traditional soil cultivation system. Soil physical and chemical characteristics was performed by the Faculty of Agriculture Menoufia University, Soil Testing Laboratory according to the standard procedures of Jackson (2005) and the obtained results are presented in Table (1).

Table 1: Principal characteristics of the soil physical and chemical analysis

Experimental design

The experiment was set up in a randomized complete block design with three replications.

The area of each plot was 36 m² (4 ridges with 12 meters long and 3 m width, and the distance between plants was 15 cm. The seeds were sown on the two sides of every ridge in which one ridge was left between the plots as a guard ridge, on a clay loam soil under surface irrigation system.

Nitrogen treatments

The experiments were carried out to study the influence of three nitrogen types on the serpentine leaf miner, Liriomyza trifolii infestation, as well as pea growth parameters and components of the yield. The recommended dose of N fertilizer was (80 kg feddan^-1) was added at two equal portions during soil preparation, and at 30 days from sowing during flowering stage. Nitrogen fertilizer treatments were added in the form of urea 46% as (172 kg), ammonium nitrate 33.5% as (239 kg) and ammonium sulphate, 20.5% N as (390 kg feddan^-1), respectively and unfertilized control.While potassium sulphate, 48% K₂O was added at two equal doses during soil preparation and during filling of the fruit, meanwhile calcium super phosphate 16.5% P₂O₅ and organic manure (10 m³ feddan^-1) was added during soil preparation. Other agriculture practices for pea production were practiced as usual in the commercial production of green pod yield.

Control program

Pea seeds (Master B) were selected for uniformity by choosing those of equal size and same color and soaked in mixture of Imidacloprid + Pencycuron (Moncyrin G 37% FS ) solution for about 6 h before sowing. All pea plots were subjected to the proposed control program as presented in Table (2). Control program consisted of foliar sequential sprays of some insecticides. Insecticide applications were applied using the calibrated Hand-Held compression sprayer (Kwazar) before the foliar application, while control plot was sprayed with water.

Table 2: Applied insecticide program in pest control against L. trifolii during 2021 and 2022 winter seasons.

^*hl = 100 liter water

Inspection of leaf miner infestations:

Weekly randomized leaf samples of pea plants (20 leaves/ replicate) were taken after 15 days of sowing prolonged to the end of experiment. Each sample was kept in paper bags and transferred to the laboratory for examination under a stereomicroscope to count the number larvae of L. trifolii. The mean numbers of L. trifolii larvae among the three tested N types was statistically compared.

Recorded plant data:

Randomly sample of five plants were chosen from each plot at 45 days from seed sowing to measure the following:

1- Vegetative growth parameters: Number of leaves, and branches/ plant, plant height (cm) and plant fresh & dry weight (g).

At harvest time, green pods, and its components, as well as pod and seed quality were measured.

2- Green pod yield and its components: Number of pods per plant, average pod weight average (g), total green pod yield (ton) / feddan, pod length and width (cm), and number of seeds per pod.

Statistical analysis:  Data were statistically analyzed using SAS v 9.3 (2003, SAS Institute Inc., Cary, NC, U.S.A.) including f-test, t-test, and simple correlation. The revised least significant differences (LSD) were used for comparing means at 5% levels of probability.

RESULTS AND DISCUSSION

Influence of nitrogen types on leaf miner infestations

The combination between the types of N fertilizers and the pest control program had been an important effect on leaf miner, L. trifolii infestation on pea plants. The data summarized in Table (3) show that the three N types stated a high significant effect on leaf miner density comparing to control plot (Prob. > |T| ranged from 0.001 to 0.0001). The infestation of L. trifolii larvae was extended between 1.23 – 2.10 larvae/leaf on pea plants with the three tested fertilizers under the application of pest control program. Contrariwise, it was exceeded than 4 larvae/ leaf with the control of these three N types without pest control program. The infestation of L. trifolii larvae recorded 1-2 peaks on all tested N types in pea plants during winter season 2021 (Table 3). Under pest control program, the nitrogen application significantly affected L. trifolii larvae infestation between ammonium sulphate and both of urea and ammonium nitrate applications during the investigated season (F value equal 3.96 and LSD=0.6313). The highest population of L. trifolii larvae were recorded with urea and ammonium nitrate fertilizers (2.10 and 1.47 larvae/leaf, respectively) (Table 3). During winter season 2022, a significant effect was reported with the three nitrogen types (Table 4). In the same direction throughout the first season, larval infestation of L. trifolii on pea plants did not exceed 2 peaks throughout the second winter season 2022, but the infestation level was very low compared with that of the 1^st season. The greatest infestations of L. trifolii larvae were also recorded with urea fertilizer  (2.04 and 4.83 larvae/leaf) and ammonium nitrate (1.41 and 4.33 larvae/ leaf), followed by ammonium sulphate (1.18 and 4.58 larvae/ leaf) with and without pest control program during second season 2022, respectively (Table 4).

It was evident from the present data that the combination between ammonium sulphate or ammonium nitrate and the suggested pest control program have an important role in reducing the leaf miner, L. trifolii infestation to pea plants. The main drivers of N fertilizer in this experiment had been identified as N formulation. In the present study, the nitrogen application significantly affected L. trifolii larvae infestation between ammonium sulphate and both of urea and ammonium nitrate applications during both investigated seasons of 2021 and 2022 respectively. The obtained data was harmony with that obtained by Cruz et al. (2012) who found that the leaf miner attack was higher in common bean plants without stress nitrogen , while more leaves, and  miner attack % in bean plants under stress of nitrogen. The present results may be due to the role of N in synthesis of proteins, oils, enzymes, vitamins in plants,  specially, the nitrogen source of ammonium sulphate which contained sulphur the main constituent of amino acids, Cystine (27% S), Methionine (21%S) and Cysteine (26%S), there are mainly the building amino acids of proteins (Patra et al., 2013). However, N nutrition levels have affected the life cycle of tomato leaf miner, T. absoluta on tomato plants. (Larbat et al., 2016). Moreover, the population of leaf miner, Liriomyza sp. on bean, Phaseolus vulgaris was related with N fertilization (Rodrigues et al. 2009). Additionally, the insect-host plant interaction show that L. trifolii development increased with increasing nitrogen levels in potato leaves (Facknath and Lalljee 2005).

Table 3: Population fluctuation of Liriomyza trifolii larvae infested pea leaves as affected by different N sources and pest control program during winter season of 2021

** = Highly significant, Means signed by the same letter in the row were non-significant

Table 4: Population fluctuation of Liriomyza trifolii larvae infested pea leaves as affected by different N sources and pest control program during winter season of 2022

** = Highly significant, Means signed by the same letter in the row were non-significant

In the present study, L. trifolii larvae population was differed according to nitrogen types as presented in Tables (3, 4) where the nitrification of nitrogen was higher with urea and ammonium nitrate than in ammonium sulphate, as well as other studies reported that N₂O emissions were higher from NO₃ −based fertilizers than from urea (Dobbie and Smith, 2003; Kuikman et al., 2006 and Jones et al., 2007). This may be due to the role of nitrogen on the syntheses of protein and amino acids that play an important role in leaf miner development (Facknath and Lalljee 2005). Additionally, in the current study, the levels of N fertilizers in pea plant may be related to N absorbance and the N stabilizers, in which, the source of nitrogen, ammonium sulphate may be more stable than other N sources in this study, so that, this treatment had been less infestation of L. trifolii larvae than others which confirmed with the research of Misselbrook et al., (2004), who found that the N₂O emissions from calcium nitrate were higher than urea. Harty et al. (2016) reported that the stability or reducing of N nitrification was established by using N stabilizer as Dicyanodiamide (DCD) or N-butyl thio-phosphoric tri-amide (NBPT) with urea and calcium nitrate, due to the bad effect on the syntheses of protein and amino acids that play an important role in leaf miner L. trifolii development. Moreover, fertilization interferes in the physiological process and growth development of pea plants, and it may be related to higher density of pea leaf miner (Nestel et al., 1994). Also, the connection between plants and herbivorous insects is typically influenced by the mineral nutrients' compliance because nutrients change the chemical composition, morphology, anatomy, and phenology of plants (Uesugi, 2015).

Effects on growth and yield characteristics:

The influence of these three tested types of nitrogen on pea growth characters illustrated that  no significant effect was statistically detected on the plant height and the mean numbers of branches per plant with the three treatments (38.67, 43.26 and 47.59 cm) for plant height and 2.17, 3.33 and 2.67 branches per plant during season 2021. However, a significant effect between these tested nitrogen fertilizers for the numbers of leaves per plant (Table 5). Contrariwise, no significant differences between all types of fertilizers and pest control treatment throughout the first season 2021.Concerning the effect of different methods of N applications, the present data reported that , the lowest value of plant weight was recorded with the treatment of  urea for two additions (185.0, 40.0 g/ 5 fresh and dry plants, respectively) while the highest  value was found with ammonium nitrate during winter season 2021 (330.0, 61.3 g/ 5 fresh and dry plants, respectively).

Data in Table (5) reported the interaction between the nitrogen fertilizers and the pea pods and seeds in the 1^st season, where there were no significant effects on the length and diameter of pods and the mean numbers of seeds/ pod, where the pod length and diameter were ranged between 7.67 – 8.83 cm and 4.22 – 5.67 cm, respectively. The mean numbers of seeds per pod was ranged from 6.83 – 8.00 seeds/pod, and the highest pod parameters were recorded with ammonium sulphate applications (Table 5). A significant effect was noticed between urea & ammonium sulphate or ammonium nitrate treatments on the numbers of pods per plant and the weight of these pods through the first season. The highest number of pods per plant was 17.00 followed by 12.33 and 11.67 with ammonium nitrate, ammonium sulphate and followed by urea applications, respectively. The same trend was stated for the weight of pod with all treatments at the winter season of 2021(Table 5).

Regarding to the obtained data through the winter season 2022 Table (6), a similar trend was noticed for impacting of N types on growth and yield characteristics of pea plant. No significant effects were recorded on the plant height, no. of branches per plant, length & diameter of pods, no. of seeds per pod and the weight of 5 dry plants during season 2022 with an average ranged from 39.97- 48.66 cm, 2.36-3.97 mean numbers/plant, 7.27-8.77 cm, 3.85-5.60 cm, 5.04-7.93 mean numbers/ pod and 51.67-87.92 g., respectively through winter season 2022. On the other direction, there was  significant effects for  nitrogen types on the mean numbers of leaves, mean numbers of pods per plant, the weight of pod and the weight of 5 fresh plants with average between 17.10- 28.10 mean numbers/ plant, 8.62-16.93 mean numbers/plant, 3.6-7.18 g. and 116.69- 356.67 g., respectively. Additionally, no significant differences were recorded between fertilizer types and pest control treatment throughout the 2^nd season 2022 (Table 6).

Table 5: Effect of three nitrogen sources on the vegetative growth and yield of pea plant, Pisum sativum under pest control program during winter season of 2021.

A*. Ammonium

Table 6: Effect of three nitrogen sources on the vegetative growth and yield of pea plant, Pisum sativum under pest control program during winter season of 2022.

** = Highly significant             * = Significant            NS= non-significant

Overall means was signed by the same lowercase letter in the same column were non-significant

Accordingly, the application of nitrogen sources were encouraged the pea vegetative growth. This increase of N fertilizers may be promoting the pea growth as well as cell division and elongation. Application of nitrogen fertilizers in form of ammonium sulphate recorded the highest values of the tested pea growth throughout both two tested seasons, this growth development can be ascribed to sulfur which enhance the plant growth as reported by Elmar (2001).

The obtained data resulted different growth and vegetative characters with the used formulation of nitrogen, which may be due to the positive effects of nitrogen fertilizer in photosynthesis, growth, stress resistance, development and oxidative-stress signaling. These results are in harmony with those reported by Abd El- Hakim (2006) and Abd El- Naem and Abd El- Hakim (2009) on some legumes.

The present study reported that ammonium sulphate was the suitable source of nitrogen compared with other nitrogen sources, and this may be due to that ammonium sulphate contains sulphur, which is a component of succinyl Co-A, a component of chlorophyll in leaves, and similar to the study of Ralsool et al. (2013) who found that these components are accelerated photosynthesis, which eventually encourages vegetative growth.

As shown in Figure (1) nitrogen fertilizer types have a substantial impact on pea production. During the two investigated seasons, 2021 and 2022, both ammonium sulphate and ammonium nitrate applications yielded the highest production of pea crop (3.345 and 3.435 tones/ feddan during 2021 and 3.418 and 3.508 tones/ feddan during 2022, respectively) with significant differences  to the lowest production (2.633 and 2.706 tones/ feddan throughout 2021 and 2022, respectively) after fertilized by urea under pest control program (Figure 1) (Prob. > |F|, F value equal 25.23 & 20.00 and LSD= 302.91 & 340.20 during 2021 and 2022, respectively.

** = Highly significant,  * = Significant,  NS= non-significant ,

Means was followed by the same lowercase and uppercase letters in the same season were non-significant

Figure 1: Pea production under pest control program during winter seasons of 2021 and 2022.

Fertilization with the selected nitrogen sources can increase pea crop yields compared with no. N fertilization with significant difference between them except with urea application which reported no significant effect under pest control program against leaf miner, L. trifolii infestations. Without utilizing a pest control program, the treatments with ammonium nitrate, urea and ammonium sulphate recorded 2.82, 2.25, and 1.8 tons per feddan during the 1^st season and 2.893, 2.323, and 1.873 tons per feddan during the 2^nd season, respectively (Figure 1).

Under the pest control program, nitrogen fertilizations have the potential to increase pea yields in comparison to no pest control program. These results are in harmony with those obtained by Sainju et al. (2019). Moreover, Metwaly (2018) found that the N fertilization sources (ammonium nitrate, ammonium sulphate, calcium nitrate and urea) significantly affect growth and yield of spinach plants. Also, El Mantawy (2017) reported that the N sources improved the sunflower production. Contrariwise, Muhammad et al. (2007) found that no significant improvement in seed yield of canola, Brassica napus. In the present study, ammonium sulphate and ammonium nitrate produced the highest pea pods compared with urea application, which are in harmony with those conducted by Malik et al. (1996) who stated that ammonium sulphate produced more weight seeds of sunflower crop than other N treatments compared with urea application. These differences in pea vegetative growth and yield may be due to exceed the accumulation of chlorophyll pigments in plants (El Mantawy, 2017). High N levels in soil increases leaf content of amino acids and proteins, as well as improving vegetative growth, delaying maturity and lignification of plant tissues, and making the plant more attractive to insect herbivores (Coqueret et al., 2017). Also, application of N in form of ammonium sulphate was exceeded pea growth, this growth development can be ascribed to sulfur which enhance the plant growth as reported by Elmar (2001). So that, ammonium sulphate was more suitable to produce pea crop in the present work.

Correlation matrix between L. trifolii larvae with N applications and yield

Regarding to the relationship between pea yield, N Applications and L. trifolii larvae infestation, Table (7) statistical analysis reported that there is a significant positive relation between N applications and population of L. trifolii larvae on pea plant in which the increasing of N applications will increase pest abundance during the two investigated seasons of 2021 and 2022. The correlation coefficient (r) recorded its highest value (0.96) with the probability 0.01 in case of N applications and L. trifolii infestation. On the other direction, the relationship between L. trifolii population (Larvae) and pea yield was significantly negative during both two tested seasons of 2021 and 2022 recording ( - 0.93 and 0.02) in correlation coefficient value and probability, respectively, moreover, the increasing of L. trifolii population will decrease the pea production through two tested seasons (Table 7).

Table 7: Mean correlation matrix between L. trifolii larvae with N applications and yield during two winter seasons of 2021 and 2022

r value = correlation coefficient                    Prob. = Probability