The Influence of Fertilization on Pomegranate Susceptibility to Infestation by Ectomyelois ceratoniae

ABSTRACT

Carob moth, Ectomyelois ceratoniae Zell (Lep.: Pyralidae), is the most important pest in Iranian pomegranate orchards and imposes tremendous annual economic loss to the pomegranate producers. A two-year study was conducted to investigate the effects of the utilization of different fertilizers on the carob moth infestation of pomegranate cv. ‘Bajestani’. Fertilizer programs significantly affected fruit infestation. The highest infestation rate was recorded in unfertilized plants (38.07%, 31.44% in the first and the second years, respectively). Among different treatments, chemical and humic fulvic-containing fertilizers proved to be the most efficient treatments for controlling carob moth. A Significant correlation was observed between carob moth infestation and various fruit and tree-related attributes. The peel hardness and its hardness-associated nutrients (Ca, K and Si), as well as total phenolic content of fruit juice, were among the factors that showed a high negative correlation with carob moth infestation. According to our results, a combination of organic and Si, Ca, and K-containing fertilizers has great practical potential in the management of pomegranate orchards to reduce the carob moth damages.

KEYWORDS:

• Pomegranate
• fertilizers
• fruit infestation
• carob moth
• correlation

Introduction

Pomegranate (Punica granatum L.) is a subtropical fruit-bearing tree, which is considered as a moderately resistant plant to different environmental stresses. This fruit tree is a good option for the lands in the border deserts, the regions, which are uncultivable for the most of crops (Zarei et al., 2009, 2016). Besides its relatively low expectation, the fruit is highly nutritious and native consumers traditionally have been using it as a medicinal fruit, a fact which is confirmed by the recent pharmacological investigations (Sreekumar et al., 2014).

During their life cycle, plant species are encountered with different biotic and abiotic stresses, which negatively affect their growth and development and ultimately reduce plant performance (Faghih et al., 2017, 2019). Pest damages are among the most important factors, which reduce plant yield and waste their products. It is estimated that insect pests are responsible for 20–40% of crop yield reduction worldwide (Douglas, 2018). Pomegranate is attacked by about 45 species of insects (Mir et al., 2012). The pomegranate carob moth [Ectomyelois ceratoniae (Lep.: Pyralidae)] is the most injurious pest in Iran, as the main center of origin and diversity of this fruit crop. It is reported that this pest reduces about 30% to 80% of the annual pomegranate production in Iran (Azqandi et al., 2015). The pest is also a major constraint for other pomegranate-producing countries including Turkey, Tunisia, Iraq, and Algeria (Azqandi et al., 2015; Demirel, 2016). The primary damage of carob moth will provide the entry site for other insects and microorganisms to the infested fruit, making the fruits more susceptible to the secondary damages. Therefore, there are further attacks of other microorganisms on the infested fruits by E. ceratoniae, bringing about serious commercial losses to farmers. The eggs of carob moth are placed in the middle of the fruit’s stamens, and the larva feeds from the internal parts of the fruit; therefore, the chemical spraying has usually failed to control this pest’s population. In addition, there is a growing consensus that the current trend of insecticide utilization is not coincident with sustainable agriculture (Douglas, 2018). Increasing insecticide resistance, high cost for formulating new chemicals, environmental toxicity, and human health issues are among the main concerns regarding insecticide application. Therefore, searching for alternative strategies to control insect crop pests with the aim of sustainable intensification of food production is getting priority in recent years (Douglas, 2018).

Proper orchard sanitation such as destroying the remnants of the infested fruits, elimination of fruit stamens, mating disruption, mass trapping, bagging fruits with fine organza mesh as well as tree strengthening are among the main effective ways that have been previously tried to control the carob moth (Mamay and Dağ, 2016; Mamay et al., 2017, 2016; Olyaie Torshiz et al., 2017; Sheikhali et al., 2009).

Soil physical and chemical properties as well as its active biological status have a significant impact on plant tolerance to different pests and diseases (Altieri and Nicholls, 2003; Azemi-Ardakani, et al., 2020). Different fertilizers are used in agriculture to improve crop yield and quality. Chemical fertilizers are among the most widespread ones, which are routinely used according to the nutritional needs of each plant. There are also different types of organic fertilizers such as animal manure and vermicompost that are utilized mainly as soil amendment since they are nutritive to the plants and contain highly different ingredients.

Plant nutritional status plays a major role in plant tolerance to different unfavorable conditions. The application of nutrients proved to be an efficient method that can mitigate the biotic stresses of the host plant against herbivory (Ramirez-Godoy, et al., 2018). The effectiveness of proper fertilization for reducing pest damages is reported in several investigations (Bala et al., 2018; Dara, 2019; El-Nabawy et al., 2016; Rashid et al., 2016; Singh and Sood, 2017). In most of the previous studies, plant responses to the nutritional status have been investigated based on the regular fertilization systems. On the other hand, the impacts of single application of different fertilizers have been investigated on the crop infestation (Sha et al., 2017; Yang et al., 2017). Most of the present knowledge regarding the relationship between crop nutrition and pest incidence is achieved from investigations in which the effects of organic and/or chemical fertilizers were compared against specific pest populations (Altieri and Nicholls, 2003). The combined effects of fertilizers according to the nutritional programs have received minor attention (Syrovy and Prassad, 2010). However, we believe that proper nutritional programs based on the plant needs during the growing season can be more effective than their individual application. In addition, there is no investigation regarding the effects of proper fertilizers on the carob moth infestation. Moreover, there are no data regarding the correlation between physicochemical attributes of pomegranate fruit and the rate of carob moth infestation.

Therefore, in the present study, the effects of utilizing different fertilizers were investigated based on the appropriate nutritional programs of pomegranate. In other words, it aimed to investigate the efficiency of combined nutritional programs for controlling carob moth infestation in pomegranate trees and to compare this type of fertilization with their individual applications.

Materials and Methods

Plant Materials, Experiment Site, and Treatments

The experiment was carried out on six-year-old pomegranate trees cv. ‘Bajestani,’ which is one of the most popular pomegranate cultivars in Iran (Figure 1). The trees have been cultured in sandy loam soil (Supplementary Table 1) with a distance of 4 × 5 m and have been trained with three main trunks under a drip irrigation system. The commercial pomegranate orchard was located at Khalilabad (35°15ʹ21ʹ’N latitude 58°17ʹ11ʹ’E longitude and 975 m.a.s.l) in Khorasan-Razavi Province of Iran. The mean annual precipitation of the region is 190 mm and the mean temperature is 17.5°C. The investigation was carried out during two successive growing seasons, 2014 and 2015.

Figure 1. Pomegranate fruit cv. ‘Bajestani’ (a); the infested fruits with carob moth (b), Infested fruits on the tree (c), and those are dropped (d); carob moth larvae (different instars) and pupae (e), and adult insect pest (f)

Treatments

The current study investigated the individual and combinational effects of different chemical, organic, and biological fertilizers on the infestation rate of carob moth. The fertilizers included chemical (Ch), humic-fulvic (Hf), biological (B), cow manure (Cm), vermicompost (V), and a combination of chemical and potassium silicate (ChSi). The chemical (Ch) and humic-fulvic (Hf) treatments were applied based on the nutritional program of pomegranate (macro and micronutrients, see Tables 1 and 2). Besides humic acid and fulvic acid, the Hf fertilizer contained a variety of macro (N, P, K, and Ca) and micro (Zn, B, Cu, Fe, and Mn) nutrients. This fertilizer was applied according to the major nutritional needs of each developmental stage. For example, most of the NPK containing fertilizers were provided in a combination of winter base dressing and through fertigation, while micro-elements containing fertilizers were sprayed during the spring. The Hf treatment also contained nano fulvic and humi power Ca nanofulvic fertilizers, which had various nutrients in the form of nanoparticles and were applied in the orchards during flowering, fruit set, and its development, as well as in the postharvest period. The biologic treatment was applied through soil, fertigation, and spraying according to the nutritional needs of pomegranate (Table 3). Characteristics of the fertilizers are presented in Supplementary Table 2.

Table 1. Different types of chemical fertilizers and conditions to be used

Stage	Chemical fertilizer	Dose	Type of application	Time	Growth stage
1	P (super phosphate triple)	558 g/tree	Sub-soil placement	February	Winter sleep
2	Urea- Co(NH2)2 −1	150 g/tree	Sub-soil placement	April	Start of leaves vegetative growth
3	Boric acid Zn N	2/1000 2/1000 3/1000	Foliar spray	April	Swelling buds
4	Humipower(no fulvic)	2/1000	Foliar spray	May	Post -flowering
5	(solupotasse) K(1)	80 g/tree	Irrigation	May	Start of flowering
6	Humipower Ca no fulvic(1)	2/1000	Foliar spray	June	Start of fruit set
7	Urea- Co(NH2)2 −2	200 g/tree	Irrigation	June	fruiting
8	(solupotasse) K(2)	80 g/tree	Irrigation	July	fruiting
9	(solupotasse) K(3)	80 g/tree	Irrigation	August	fruiting
10	Humipower Ca no fulvic(2)	2/1000	Foliar spray	August	fruiting
11	Urea- Co(NH2)2 −3	60 g/tree	Irrigation	July	fruiting
12	Urea- Co(NH2)2 −4	70g/tree	Irrigation	September	fruiting
13	Boric acid Zn N	2/1000 2/1000 5/1000	Foliar spray	November	Post-harvest
14	Urea- Co(NH2)2 −5	170 g/tree	Irrigation	November	Post-harvest

Table 2. Humic-fulvic (Hf) nutrition program used in this investigation

Stage	Chemical fertilizer	Dose	Type of application	Time	Growth stage
1	P (super phosphate triple)	280 g/tree	Sub-soil placement	February	Winter sleep
2	Humipower (N,P,K)	2 kg/tree	Sub-soil placement	February	Winter sleep
3	Urea- Co(NH2)2	150 g/tree	Sub-soil placement	April	Start of leaves vegetative growth
4	Nanofulvic fruit set (N, Boric acid, Zn)	2/1000	Foliar spray	April	Swelling buds
5	Humi power nanofulvic	2/1000	Foliar spray	May	Post -flowering
6	K plus(1)	40 g/tree	Irrigation	May	Start of flowering
7	Humi power Ca nanofulvic (1)	2/1000	Foliar spray	June	Start of fruit set
8	N plus(2)	30 g/tree	Irrigation	June	fruiting
9	N plus(3)	30 g/tree	Irrigation	July	fruiting
10	K plus(2)	40 g/tree	Irrigation	July	fruiting
11	K plus(3)	40 g/tree	Irrigation	August	fruiting
12	Humi power Ca nanofulvic (2)	2/1000	Foliar spray	August	fruiting
13	N plus(4)	30 g/tree	Irrigation	September	fruiting
14	N plus(5)	85 g/tree	Irrigation	November	Post-harvest
15	Nano fulvic fruit set (N, Boric acid, Zn)	2/1000	Foliar spray	November	Post-harvest

Table 3. Type of biological (B) fertilizers and their application programs

Biologic fertilizer	Trade name	Bacteria	Application time
			First	Second	Third
Biologic N	Azetobarvar	Azetobacter winland: (04)	First leave appearance	First fruiting	After 20 days
Biologic P	Phosphate barvar	Pantoa glomerance (05) Pseudomonas putida (p13)	Flower bud swelling	–	–
Biologic k	Pottabarvar	Pseudomonas vancouverensis Pseudomonas coreensis	After petal falling	First fruiting	After 20 days

Two types of organic fertilizers were applied in this study. Cow manure (Cm) (25 kg/tree) was used in the first year and vermicompost (V) (5 kg/tree) in both years. As the cow manure has high stability in the soil and local gardeners apply this fertilizer every couple of years, we did not use this fertilizer in the second year of the experiment. The organic fertilizers were applied in sub-soil placement method in the winter (February). To do this, four 30 cm depth holes were made using a drill 50 cm from the trunk. According to the manufacturing company, 1 l of bio-fertilizers was added to the organic fertilizers (250 ml in each hole). Trees were irrigated every four days by four drippers per tree. All of the individual treatments were given to the soil. The fertilization was done through soil, irrigation, and spraying in the case of combined treatments. The characterizations of fertilizers are presented in Supplementary Table 1.

Trial Evaluation

The fruits of each tree were harvested separately. Pomegranate fruit moth infestation (%) was expressed as a fraction of the infested fruits (both dropped and retained) on the tree (Figure 1) over the total number of fruits per tree (Sobhani et al., 2015).

Weight-related traits were measured using a digital balance with 0.01 g accuracy. Fruit peel thickness was evaluated using a texture analyzer (Testometric Co. M350-10CT) according to the method described by Zarei et al. (2013). In each tree, the percentage of sunburned and cracked fruits was calculated by dividing the number of sunburned and cracked fruits by the total number of fruits per tree. Fruit peel thickness was measured using a digital caliper. A minimum of five fruits from each treatment was immersed in a graduated cylinder with a certain volume of water to estimate their size. The total phenolic content of fruit peel and juice was estimated using Folin–Ciocalteu reagent according to the method described by Velioglu et al. (1998).

Total arils of fruits were squeezed to obtain the fruit juice. The juiciness of fruit was determined by dividing the amount of total aril’s juice by the total fruit weight. Fruit juice was used for total soluble solid (TSS), titratable acidity (TA), and ascorbic acid (AA) measurement. TSS was estimated by a refractometer (pocket PAL-1 ATAGO Corporation, Tokyo, Japan) in Brix. To estimate the TA, fruit juice was titrated with 0.10 N NaOH to pH 8.10 and the data were expressed as the amount of citric acid (g l⁻¹) as the main component of pomegranate organic acid (Zarei, 2017). Taste index was calculated based on the TSS and TA factors according to Martinez et al. (2006).

Fruit peel and tree leaves were dried in an oven at 70°C and their ashes were used to measure different elements. The Kjeldahl method was used for nitrogen (N) determination. The phosphorous (P) content was measured spectrophotometrically using the molybdovanadate yellow color method. An atomic absorption spectrophotometer (Shimadzu Co., Kyoto, Japan) was used to measure Iron (Fe), Magnesium (Mg), Zinc (Zn), Silicon (Si), Calcium (Ca), and potassium (K).

Experimental Design and Data Analysis

The experiment was carried out in a completely randomized block design (CRBD) with eight treatments and six replicates for each treatment (1 replication = 1 tree). To evaluate the effects of different treatments on the fruit characteristics, the data set was subjected to the analysis of variance (ANOVA) using SAS software and where the differences between group means were significant, values were compared using Duncan Multiple Range Test (DMRT). The level of significance was set at 5%. Bivariate correlation analysis was carried out by SPSS statistics software ver. 19 (IBM Corp, 2010) to estimate the relationship intensity between carob moth infestation and other fruit and tree characteristics.

Results and Discussion

Carob Moth Infestation

Pomegranate fruit moth infestation was significantly affected by the application of different fertilizer treatments. Control plants, which did not receive any fertilizers, had the highest percentage of infested fruits, 31.44%, 38.06% in the first and the second year of the experiment, respectively (Table 4). Positive effects of organic matters and proper nutritional treatments on the reduction of pest infestation were reported in other crops (Altieri and Nicholls, 2003; Bala et al., 2018; Dara, 2019; Singh and Sood, 2017). It is well established that healthy plants are better able to withstand the pest attacks than unhealthy ones. The nutritional status of the plant is one of the main factors contributing to the plant health. It is well documented that balance in nutrients efficiently reduces the pest infestation (Phelan et al., 1995). Lower corn borer egg-laying (Ostrinia nubilalis) was reported in the corn growing in the organic-matter containing soils than the conventionally managed soil (Phelan et al., 1995). It is reported that the density of colorado potato beetle was decreased by the application of manure to the soil (Alyokhin et al., 2005). Similar results were reported about fruit crop species (Schumann et al., 2010; Syrovy and Prassad, 2010). According to Schumann et al. (2010), a balance between macro and micronutrients in Citrus decreases pest injury. Syrovy and Prassad (2010) expressed that culture media, which were amended with organic materials, provided a better condition for the plant through buffering soil pH, moisture, and mineral nutrients. Though the importance of gradual nutrition in this research was more than the content of soil organic matter. It is reported that Si application efficiently reduced the population of Aleurocanthus woglumi (Hemiptera: Aleyrodidae) in tangerine trees (Vieira et al., 2016). Moreover, both foliar and soil application of Ca, K, and Si reduced the population of Asian citrus psyllid, Diaphorina citri (the main vector of huanglongbing disease), on Tahiti lime trees and caused a reduction in the huanglongbing disease (Ramírez-Godoy et al., 2018).

Table 4. Mean comparison of fruit infestation (%) for different nutritional treatments in two consecutive years

		Fertilizer treatments
		Vermicompost (V)	Cow Manure (Cm)	Biologic (B)	Chemical (Ch)	Chemical + Potassium silicate (ChSi)	Humic,Fulvic (HF)	Humic,Fulvic + Potassium silicate (HfSi)	Ch+V	Ch+Cm	Ch+Gh	Control
Infestation (%)	First year	30.30^a	28.92 ^ab	27.59^b	19.54 ^cd	18.75 ^cd	18.35 ^cd	17.62^d	18.05 ^cd	20.54 ^c	17.66^d	31.44^a
	Second year	37.3 ^a	35.61^a	35.48^a	25.13^b	28.67^b	24.39^b	24.35^b	26.21^b	24.9^b	25.75^b	38.06^a

Values with the same letter in each row have no significant differences at (P ≤ 0.01) probability following DMRT.

Ch+Gh treatment is the chemical treatment program with 2 kg granule humic in the winter.

ChSi and HfSi treatment is combined treatment Ch and Hf with silicate potassium spray (2/1000 dose) in 2stage: 1-Swelling flower buds 2-fruiting

Due to the occurring of a late spring cold in the region where the study was conducted and subsequent fruit abscission, the overall fruit infestation with carob moth was lower in the first year of the experiment in all treatments. The humic fulvic + potassium silicate (HfSi) treatment resulted in the lowest fruit infestation in both seasons (17.62% and 24.35% in the first and second seasons, respectively) (Table 4), reaching a 13.82 and 13.71 points decrease in carob moth infestation compared to the control plants in the first and second seasons, respectively. In addition to their positive impacts on the physical, chemical and microbiological properties of the soils that improve the efficiency and uptake of available nutrients and primary metabolites, Hf substances are also able to increase plant tolerance to different unfavorable conditions mainly through stimulating the antioxidant enzymes, activating the secondary metabolite pathways such as phenylpropanoid and shikimic acid, and influencing the Ca⁺² cell signaling (Canellas et al., 2015). Therefore, it seems that providing a combination of different elements during the growing season will be beneficial to enhance fruit tolerance against the carob moth.

Si-containing treatments showed to be the most efficient fertilizer for conferring carob moth tolerance in pomegranate fruit. Previous reports indicated that some of the nutrients are highly efficient against insect pest damages in different plant species (Alhousari and Greger, 2018; Yang et al., 2017). It is well documented that Si can efficiently improve plant resistance to the various herbivorous insects (Alhousari and Greger, 2018; Massey and Hartley, 2009; Nikpay and Nejadian, 2014; Ramírez-Godoy et al., 2018; Vieira et al., 2016; Yang et al., 2017; Ye et al., 2013). In fact, this element can reduce the insect pest incident both directly and indirectly. The direct defense system is associated with mechanical or physical barrier development that interferes with insect feeding behaviors such as cell wall intensification and/or secondary metabolite production (e.g., phenols and lignin) through its regulatory effects on different enzymes and metabolic pathways (Alhousari and Greger, 2018). The indirect defense systems are mediated by plant volatile compounds (e.g., jasmonate), which are released from host plants in response to the insect pest (Ye et al., 2013). These volatiles may reduce the pest damages by repelling the insect pest or attracting its parasites and predators. Although the results of the current study showed that fruit peel intensification contributes to higher tolerance to carob moth, the indirect defense system may also be involved in this phenomenon. Thus, it deserves to be explored in future investigations.

The combined application of chemical (Ch) fertilizers with granule humic (Gh) also resulted in a lower infestation of fruits (17.66% and 25.75% in the first and the second years of experiment, respectively) and showed no significant difference with HfSi treated plants in both years. Although humic fulvic-containing treatments showed significantly lower carob moth infestation in the first season, there were no significant differences among different chemical and humic fulvic-containing fertilizers in the second year of the experiment (Table 4). There were no statistically significant differences between carob moth in the control plants and those treated with sole CM and V fertilizers. In comparison with the chemical fertilizers, most of the organic fertilizers are relatively weaker sources of essential nutrients. Thus, these types of fertilizers are routinely used for their indirect effects on plant growth by amending plant bed and soil structure. On the other hand, these types of fertilizers are able to improve the porosity of the root surrounding areas, increase its cation exchange capacity (CEC), and have beneficial effects on the soil microorganism communities. Moreover, most of the organic fertilizers need relatively more time than their chemical counterparts to have their ultimate influence on crop performance. However, the combined application of these organic fertilizers with chemical ones resulted in a lower fruit infestation in both years. These measurements support the idea that the organic soil amendments are able to improve the uptake of chemical fertilizers and are highly efficient in increasing their effects. Among the single treatments, the application of chemical fertilizers resulted in a significantly lower fruit infestation with no statistically significant differences with HfSi and Ch+Gh treated plants in both studied years. Therefore, the results indicated that the proper application of chemical fertilizers during the growing season at different stages of plant development significantly reduced the carob moth losses in pomegranate fruits. These observations emphasize the important role of plant nutritional status to cope with the biotic environmental stresses which can be used practically to reduce the carob moth infestation damages and increase the yield and quality of pomegranate fruit.

Correlation Analysis

To determine the main factors affecting the carob moth damages on the pomegranate fruit and to estimate the percentages of fruit infestation with some of the important fruit and tree characteristics, the Pearson correlation was employed. The results of simple bivariate correlation showed that there are significant positive and negative correlations between carob moth infestation and different physic-chemical attributes of fruit and tree (Table 5). Among these attributes, some showed a significant correlation in both years and some in one year. However, we only considered those traits that showed statistically significant correlations in both years of study. Among the different fruit physical properties studied in this investigation, fruit peel hardness, both at crown and fruit diameter (equator), showed a significant negative correlation with carob moth infestation. Peel hardness at fruit crown showed the highest negative correlation (−0.55 and −0.63 for the first and second years, respectively). Pomegranate peel hardness, fruit taste, peel thickness, and color play a role in reducing carob moth infestation (Mamay et al., 2014). Therefore, it seems that carob moth activity is negatively correlated with fruit peel hardness in pomegranate, and this parameter significantly affects the carob moth progress on the way of fruit piercing. However, despite the common conceptualization of the role of fruit peel thickness on the carob moth infestation, this factor had no significant correlation with carob moth infestation in the second year. Therefore, it could be concluded that peel texture is a more determining factor for reducing the carob moth infestation than its thickness. Other physical properties of fruit including weight, cracking, and sun-burning rate had no significant correlation with carob moth infestation. However, the volume of fruit showed a high negative correlation in the second year of the experiment, suggesting a possible delayed effect. In addition, our results indicated that the total phenolic compound of fruit peel had no significant correlation with carob moth infestation in both seasons. As the larva of carob moth feeds from the inner layer of pomegranate fruit skin, the total phenolic compounds of fruit peel may have not been directly involved in the carob moth infestation. However, the total phenolic content of fruit juices showed significant negative correlations with carob moth infestation in two consecutive years (r = −0.32 and r = −0.38, respectively). The production of high levels of phenolic compounds is one of the main defensive systems developed in terrestrial plants to cope with the unfavorable conditions, especially the biotic stresses (herbivory). Therefore, it seems that providing the essential fertilizers enables plants to allocate sufficient substrates for the metabolic pathways, such as phenylpropanoid, which are actively involved in the production of phenolic compounds and resistance to different stresses. It is well documented that plants are able to protect themselves from pest damages by emitting different volatile compounds as a highly evolved chemical language (Villamar-Torres et al., 2018). Some of the volatiles may be derived from the phenylpropanoid pathway, as the main pathway for producing phenolic compounds. Therefore, phenolic compounds of aril juice may act as a barrier for insect attack to the host plants. The taste index was another fruit juice-related trait that showed a high negative correlation with carob moth infestation in both seasons (r = 0–0.37 and r = −0.40, respectively). This factor is a derivative of TSS and TA content of the fruit juice. Moreover, previous reports indicated that TA is the main determining factor for the sensory taste of pomegranate fruit juice (Zarei, 2017). Our finding indicated that carob moth infestation was negatively correlated with TA in both years of study. On the other hand, the higher the fruit acidity is, the lower the carob moth infestation would be. In accordance with our findings, the results of the previous study indicated that Ectomyelois ceratoniae prefers sweet-tasted cultivar and its infestation is lower on the sour-tasted cultivars compared with the sweet-tasted ones (Mamay et al., 2014).

Table 5. Correlation index between the percent of carob moth infestation and some of the fruit-related attributes

Trait	Fruit infestation
		First year	Second year	Significant correlation in both years
Fruit properties	Peel thickness	−0.25*	0.20^ns
	Peel hardness (fruit diameter equator)	−0.32 **	−0.30**	**
	Peel hardness (crown)	−0.55**	−0.63**	**
	Weight	0.06 ^ns	−0.008 ^ns
	Volume (cm3)	−0.26 ^ns	−0.56 **	Possible delayed effect
	Cracking	−0.11 ^ns	−0.20^ns
	Sunburning	0.20 ^ns	0.20^ns
	Total phenolic content (fruit peel)	0.10 ^ns	−0.07^ns
Fruit juice	Juiciness	−0.08 ^ns	−0.54**
	TSS	0.23 ^ns	−0.48**	Possible delayed effect
	Sugar	−0.12 ^ns	−0.16^ns
	Total phenolic content	−0.32**	−0.38**	**
	TA	−0.38 **	−0.19*	*
	Taste index	−0.37 **	−0.40**	**
Fruit peel nutrients	Ca % (fruit peel)	−0.40**	−0.59**	**
	Si % (fruit peel)	−0.35**	−0.17*	*
	K % (fruit peel)	−0.49 **	−0.57 **	**
Tree leaf nutrients	N (leaf)	−0.25 *	−0.58^ns
	P (leaf)	−0.04 ^ns	−0.05^ns
	K (leaf)	−0.02 ^ns	−0.08^ns
	Ca (leaf)	−0.34 **	−0.31*	*
	Mg (leaf)	0.15 ^ns	−0.22^ns
	Fe (leaf)	−0.45 **	−0.28*	*
	Zn (leaf)	0.20 ^ns	−0.29*	Possible delayed effect
Tree yield related trait	Tree yield	−0.16^ns	−0.66**	Possible delayed effect
	Fruit numbers	−0.06 ^ns	−0.59**	Possible delayed effect
	Fruit drop %	0.41 **	0.59**	**
	Total tree flowers	−0.42 **	−0.39**	**
	Fertile flowers	−0.25*	−0.35**	*

*: statistically significant at 1% probability; **: statistically significant at 5% probability.

A high negative correlation was also found between carob moth infestations and fruit peel nutrients. Ca content of fruit peel had a significant high negative correlation (P ≤ 0.01) with carob moth infestation in both studied seasons (−0.40 and −0.59, respectively). The Ca content of tree leaves also had a negative correlation (P ≤ 0.01) with carob moth infestation in both years (r = −0.34 and r = −0.31 for the first and the second years, respectively). The same trend was also observed in the case of K content (r = −0.49 and r = −0.57, respectively) of fruit peel. However, there was no significant correlation between K content of the tree leaves and carob moth infestation. Fe content of tree leaves was also negatively correlated (P ≤ 0.01) with carob moth infestation in both seasons. Si was another nutrient that showed a significant negative correlation with carob moth infestation in the first year of the experiment, but it showed no significant correlation in the second year.

It is now well established that healthy/vigorous plants are better able to cope with various biotic and abiotic stresses (Altieri and Nicholls, 2003). In addition to producing a healthier plant, there are other ways that some of the nutrients specifically help plants resist more against the invasion of organisms. Ca is a typical essential nutrient that contributes to the plant cell wall stabilization as well as membrane integrity and has been used extensively for strengthening various plant tissues (Hoseinbeigi et al., 2019; Korkmaz et al., 2016). The previous report indicated that Ca fertilization significantly reduced the food preference of red palm weevil (Rhynchophorus ferrugineus Oliv.) and resulted in the lower infestation in different cultivars of date palm (Phoenix dactylifera) and its wild relative, the Mazari palm (Nannorrhops ritchiana) (Farazmand, 2002). Besides its structural role, this nutrient has great regulatory importance and acts as a secondary messenger in the cytoplasm as well as counter-ion for organic and inorganic anions in vacuoles and ameliorates stresses (Tuteja and Mahajan, 2007).

Therefore, the positive effects of chemical fertilizers on lessening carob moth infestation might be slightly attributed to providing sufficient nutrients which may contribute to the tissue tightening. Our results are in the line with the previous reports indicating that Ca fertilizers were able to improve plant tolerance to different insects presumably through the reinforcement of the cell walls and tissues (Hua et al., 2015; Ramírez-Godoy et al., 2018).

Previous reports indicated that K containing fertilizers significantly reduced the pest infestation in many host plants and/or K-deficient plants showed more susceptibility to the pest infestation than those received an adequate amount of this nutrient (Holzmueller et al., 2007; Myers and Gratton, 2006; Perrenoud, 1990; Sarwar, 2012; Sudhakar et al., 1998; Wang et al., 2013). In contrast to the Ca nutrient, which is structurally important, K ion has a regulatory function and activates different enzymes, facilitates translocation of other compounds including Ca and amino acids, and contributes to the cell integrity through affecting the synthesis of cell wall components such as cellulose (Mengel, 2001). Thereby, K enables plants to develop stronger cell walls and improve plant defense systems against pathogen infestations.

Although Si is not considered as an essential nutrient in higher plants, it is well established that this element plays a protective role, increases the mechanical supports of plant tissues, and strengthens the cell walls through interacting with cell wall components such as cellulose, hemicellulose, and lignin (Guerriero et al., 2016).

However, among different nutrients of tree leaves that we measured in this investigation, only Ca and Fe showed a significant negative correlation (P ≤ 0.05) with carob moth infestation in both seasons.

Some of the tree yield-related attributes were also statistically correlated with carob moth infestation. The total number of flowers and the fruit drop percentage were two yield components that have been significantly correlated (P ≤ 0.01) with fruit infestation. The number of fertile flowers also showed a negative correlation with fruit infestation (P ≤ 0.05). The fruit drop percentage was the only studied factor that positively correlated with fruit infestation. In fact, fruit drop is a direct consequence of pest attack and a normal response of many plants to the pathogen invasion to reduce the subsequent damages to the tree (Botton et al., 2011; Hagemann et al., 2015). However, total flowers and fertile flowers were negatively correlated with carob moth infestation. An increase in the tree flowers can be attributed to the improvement in the nutritional status of the tree, which ultimately resulted in the higher fruit set and tree yield in the second year of the experiment. Fruit numbers and tree yield also showed a significant negative correlation with carob moth infestation in the second year.

Conclusion

Our results indicated that individual application of organic fertilizers did not affect the carob moth damages, while their combination with chemical ones, which were nutrient elements prepared and supplied gradually according to the nutritional needs of pomegranate trees during the growing season, resulted in a lower carob moth infestation. According to the observations, diversification of nutritional methods will provide the nutritional requirements of pomegranate trees and subsequently reduce the level of carob moth infestation. Moreover, the results of this investigation suggest that a combination of chemical fertilization based on the nutritional programs of pomegranate tree with organic materials can be successfully used in horticultural practices to reduce crop losses that are resulted from nutrient deficiency as well as the pomegranate fruit moth infestation. This method may have a significant practical application for controlling carob moth infestation. Therefore, combined nutrition proportionate to the needs of pomegranate trees can be considered as a strategy for the integrated pest management of carob moth. In addition, the bivariate correlation analysis revealed that there are significant correlations between some of the fruit and tree attributes that could be used in the future studies to improve the hardiness of pomegranate fruit to the carob moth. Among different evaluated traits, hardness-related traits showed to be highly effective for controlling carob moth. In general, Ca, K, and Si elements in combination with organic fertilizers are suggested as the main nutrients that could be used practically for reducing carob moth infestation in pomegranate. Possible delayed effects should be studied in further research.

Acknowledgments

This research is supported by the University of Tehran and the authors are grateful to Mr. Parhizi, the orchard man, and all employees of the orchard for their helpful cooperation during this study. Many thanks go to Mr. Goddusi, the laboratory expert at the University of Science and Culture, and Mr. Mirhajian, the manager of Sabzarsham Yazd Company for their cooperation in providing the fertilizers.

Additional information

Funding

This work was supported by the College of Agriculture Natural Resources, University of Tehran [NA].