Effect of poplar (Populus nigra) spring and autumn leaves extract on pepper (Capsicum annuum) infected with pepper mild mottle virus under greenhouse conditions

Abstract

Capsicum annuum is one of the main vegetable crops for the local market and exportation in Egypt. In this concern, pepper mild mottle virus (PMMoV) infection caused a significant decrease in Capsicum sp. yield, leading to large economic losses. An isolate of PMMoV was got from naturally infected pepper plants, exhibiting different patterns of mottling, leaf distortion, yellowing, and stunting of leaves. The virus was identified. The molecular detection of PMMoV was done using RT-PCR with specific primers designed for coat protein genes. An RT-PCR product (~ 474) bp of the coat protein gene of (PMMoV) was cloned. The target of the investigation was the effect of spring and autumn ethanol extracts of Populus nigra leaves on C. annuum seedling growth and infected C. annuum with (PMMoV) under greenhouse conditions. The experimental data showed that treated spring leaf extract of P. nigra enhanced infected C. annuum seedling growth parameters and fruit quality compared to uninfected seedlings. P. nigra spring leaf extract containing some allo-chemicals had a negative effect on uninfected seedlings. P. nigra autumn leaf extract significantly improved the growth and fruit quality of infected C. annuum seedlings compared to the control.

Introduction

Sweet pepper (C. annuum) is a member of the solanaceae vegetables. It considered a major important greenhouses yield cultivated during different seasons to convene increasing demand in Egypt. The cultivation area reached 91404 feddans. Selim, et al., (2021).

Pepper mild mottle virus (PMMoV) has only newly been identified on commercial bell pepper field in Florida, Italy, Adkins, et al., (2001). The virus is spread by mechanical means and infected seeds but cannot be transmitted by insects. It is worldwide in field grown bell, hot and ornamental pepper. It is found in pepper cultivars where production practises are typical for rapid spread of disease Secrist, (2021). As foliar symptoms can be mild, infected plants may not be noticed up till fruit symptoms are evident, outcome in spread to adjoining plants and higher yield losses Adkins, et al., (2001). (PMMoV) causes serious economic losses in pepper production in China. Han, et al., (2020) identified two PMMoV isolates (named PMMoV-ZJ1 and PMMoV-ZJ2) with decrement symptoms in a survey for viral diseases on pepper in Zhejiang province.(PMMoV) infected display dwarfing, mottling, puckering, deformed foliage, and fruits in general appeared small and malformed, and this was obvious by off-colored sunken areas. Al-Khayri, et al., (2021) found that virus causing harm damage to pepper yield.

Populus nigra known as cottonwood, poplar, and aspen deciduous trees Jansson, et al., (2010). Trees produce a large quantity of fallen leaves as a waste during autumn in Egypt's environment. P. nigra is a member of the Saliaceae Fam., which includes several species and is distributed extensively throughout the world. Bradshaw, et al., (2000). Poplar leaves are used as a boost to antimicrobials. Al-Hussaini, and Mahasneh, (2009). A lot of bioactive structures such as terpenoids and flavonoids, in addition to phenolic compounds, have been extracted from Populus sp. by Radoykova, et al., (2010); Schnitzler, et al., (2010); Dudonné, et al., (2011); Zhong, et al., (2012). In this regard, the results provided a hopeful baseline in sequence for using flavonoids from these trees as antimicrobials to control plant diseases. Zhong, et al., (2012). Meanwhile, a lot of flavonoids with the structure pinobanksin and 3,7-dimethylquercetin as well as pinocembrin were separated from the P. nigra ethanol extract by Adams, et al., (2009). On the other hand, phenolic structures with caffeic and p-coumaric additions to cinnamic were mentioned by Dudonné, et al., (2011). Previous investigations studied poplar trees' naturally occurring aromatic compounds such as salicylic acid and salicylic alcohol. Pearl, and Darling, (1971); Steele, and Ronald, (1973). Plants and their extracts have been used for medicinal purposes since ancient times. According to the World Health Organization, over 75–80 percent of the world's population uses plant medicine in some form or another. Willow bark, for example, was eaten by ancient Egyptians to ease fevers and headaches. Scientists found thousands of years later that the bark contained salicylic acid, the key element in aspirin. John Buchner discovered salicyl alcohol glucoside (Salicine) from willow bark in 1928, and Rafacle Piria called the compound salicylic acid (hereinafter SA) in 1938. according to Yusuf, et al., (2013).

The study aimed to evaluate the effect of foliar (25–50 and 100%) concentrations of spring and autumn leaves (APLE) on C. annuum seedlings and fruit parameters. As well as studying the effects of (25–50 and 100%) concentrations of (SPLE) and (APLE) foliar applications, the process took 20 and 40 days from germinating infected pepper under greenhouse conditions in the Ismailia region.

Materials And Methods

The field experimentent was carried out during the two growing seasons of 2019 and 2020, at Ismailia Governorate, Egypt, in order to investigate the effect of concentrations (%25–50 and 100) foliar application of spring Populus nigra leaves extract (SPLE) and autumn Populus nigra leaves extract (APLE) treated after 20 and 40 days from germinating under infection with (PMMoV) on enhancing resistance, growth, yield of pepper plants Capsicum annuum. The plants were cultivated on pots 30 cm in mixed loam and sand (1:1) under greenhouse conditions. Using the normal (recommended) fertilization programmer for this crop.

Tree Materials 

Collection of tree material: P. nigra spring leaf was collected in March 2019 and 2020 from trees growing in the nursery of the timber trees department at the Horticulture Research Institute Agriculture Research Center, Giza, Egypt. Senescent leaves were collected in September (2018 and 2019).The samples were dried in the electric oven at 40˚C until they reached constant weight according to Hernández-Castillo, et al., (2010). Dried material was ground by an electric mixer to find the crush forms of each sample. The powder was preserved in sterilised glass jars.

Preparation extracts samples:

The samples were air dried in the laboratory for seven days under room conditions and later in the electric oven for two days at 40˚C Raja, et al., (2019). The dried material was then pulverised using a blender (electric mixer) to obtain powder forms of each sample. The powder was collected and kept in clean and sterile conditions. Each leaf in spring and autumn, an individual dried powdered sample (500g) is laid in a 2000 ml beaker and processed by drenching (1000 ml) of ethanol solvent. Then they were enclosed with aluminium foil and put into a water bath (60˚C) and shaken to obtain homogenous solutions. After that, the samples were filtered and evaporated by a rotary evaporator (60˚C) to isolate the solvent. extract and store it in clean-capped glass bottles and reserve it in the refrigerator for ruse. Wang, et al., (1996).      

Source of virus isolates:

Several field visits were conducted to pepper plant growing areas in the Ismailia Governorate. The naturally infected pepper plants showing viral symptoms including mottling, leaf distortion, yellowing, and stunting were collected. After being collected from the field, the infected leaf samples were placed in cool boxes and stored at -80 °C for later use.

Propagation of virus isolates:

Infected leaf samples were ground in a phosphate buffer solution (pH 7.2). Infectious sap was mechanically inoculated onto Chenopodium amaranticolor. The single local lesion assay was used for biological purification of the isolate and propagated on healthy pepper plants.

Virus identification:

Host range and symptomatology:

Twenty-one plant species belonging to four families were mechanically inoculated with infectious crude sap expressed from pepper plants. The seedlings of each host species were inoculated and observed daily for symptom development, and the mechanically inoculated plants were kept under observation in insect-proof cages under the greenhouse. Three weeks later, plants were examined visually for any signs of symptom appearance. Symptomless plants were checked for virus infection by back inoculation of Chanopodium amaranticolor leaves and/or the ELISA technique by Clark, and Adams, (1977).

Different symptoms were observed on the infected pepper plants. The virus infection shows mottling, leaf distortion, yellowing, and stunting. The virus was propagated on pepper plants, which developed the same symptoms as those in naturally infected plants. The isolate of PMMoV shows different styles of symptoms on pepper hosts, such as mild mottling, mottling, yellowing, and malformation (Table 1 and Fig. 2). The incidence of PMMoV was confirmed by back inoculation with Chanopodium amaranticolor. The tested plants could be divided, according to their reactions, into two groups:

 Susceptible hosts to PMMoV.

a- Plants reacted with systemic symptoms.                                                              .
 Systemic symptoms were observed in the tested Capsicum annum L. cv. California, Capsicum fratescens L. cv. Chilli and Nicotiana clevelandii Fig. (2). Systemic symptoms, generally appear nearly 11–14 days after inoculation..

b- Plants reacted with local lesions.                                                                             .
   Virus isolate produced chlorotic local lesions on the inoculated leaves of Chanopodium amaranticolor, Ch. quinoa and necrotic local lesions on the inoculated leaves of Datura metal, Datura stramonium, Nicotiana tabacum, and Ni. glutinosa nearly 7–10 days after inoculation (Fig. 2). 

2-Unsusceptible plants.

These plant species were not susceptible to pepper infection. These plants belong to different families: Cucurbitaceae, Fabaceae, and Nicotiana arusica. Host range studies for diagnosis will usually be most useful for those infecting a relatively narrow range of plants Kaundal, et al., (2011).

The general outlook of the results in table (1) indicates that the studied isolate of PMMoV had a wide host range between members of the family Solanaceae. On the other hand, the virus infects a few species of Chenopodiaceae. PMMoV induced mottling, yellowing, and malformation symptoms in the family Solanaceae. The obtained data in table (1) confirmed the results of Wetter, et al., (1984).
 

Table (1): The reaction of different hosts to Pepper mild mottle virus.

Modes of transmission:

Mechanical transmission.              

Inoculums were prepared by homogenising infected pepper leaves with a few drops of phosphate buffer (pH 7.2) in a sterilised mortar. Leaves of host plants previously dusted with carborandum (600 mech) were rubbed with the forefinger or with a cheesecloth pad previously soaked in the inoculum. The plants were rinsed with tap water and kept in the insect proof greenhouse.

Obtainment results revealed that PMMoV was easily transmitted mechanically to indicator hosts like Chenopodium amaranticolor which showed chlorotic local lesions.. 

Insect transmission.

Two aphid species, namely, Aphis faba (scop) and Myzus persicae (sulz) were checked for their ability to transmit the isolated virus. Aphis faba (scop) and Myzus persicae (sulz) were maintained on virus- free healthy faba beans for Aphis faba (scop) and cabbage plants for Myzus persicae (sulz) and kept under insect-proof cages in the greenhouse. The aphids were starved for one hour and then transferred to feeding for a 30 minute acquisition feeding period on diseased pepper plants. At the end of the feeding period, aphids were transferred to healthy plants at a rate of 10 aphids/plant. After a 24 hour feeding period, the insects were killed by spraying all tested plants with an effective insecticide (malathion 0.2%). Symptoms and the percentage of transmission were recorded. 

Results indicated these Aphis faba (scop) and Myzus persicae (sulz) were not able to transmit the virus. None of the tested plants produced any symptoms.

Seed transmission of virus:

To study the transmission of (PMMoV) through seeds. Two hundred pepper seeds cv. California collected from previously inoculated infected peppers were sown in 20 cm sterilized pots and kept in an insect- proof greenhouse for symptom observation till three weeks after sowing, and the percentage of seed transmission was calculated.

(PMMoV) was transmitted through pepper seeds. Data showed that the percentage of seed transmission differed according to cultivar. (PMMoV) was transmitted at 38%. The result was confirmed using ELISA. 

Molecular characterization:

RNA extraction. 

RNA extraction from leaf samples was carried out using the RNeasy Plant Mini Kit (QIAGEN) according to the manufacturers’ instructions.

Primers for the coat protein gene of (PMMoV):

For the amplification of the capsid protein (CP) gene (474 bp), two pairs of specific primers (CP/s: 5′-ATGGCATACACAGTTACCAGT-3′) and (CP/a: 5′-TTAAGGAGTTGTAGCCACACGTA3′) were used in RT-PCR Çağlar et al., (2013).

One-step RT-PCR:

One-step RT-PCR reactions were carried out using the "iScript One Step qRT-PCR Kit" (BIOMATIK) in a 25 µL reaction volume. Each reaction contained 1 µL of the RNA extract (40 ng of total RNA), 12.5 µLi Green Mastermix, 1.5 µL of 10 µM of each primer, 0.5 µL of qRT-PCR Enzyme Mix, and 25 μL of nuclease-free water. Synthesis of cDNA was done at 42°C for 30 min and denaturation at 95°C for 10 min, followed by 35 cycles of 94°C for 30 sec, 50°C for 1 min, 72°C for 1 min, and a final cycle of 72°C for 10 min Velasco, et al., (2011). 5 μL of PCR products were loaded into 1% agarose gels with a 100 bp DNA ladder (BIOMATIK) and pictures were taken under UV light with a digital imaging system gel doc (Syngene Bio Imagins, IN Genius).

Analysis of RT-PCR products:

The cp genes of PMMoV collected from Ismailia was isolated using RT-PCR with specific primers. The PMMoV-cp gene had (~474) bp (Fig.3).

Experimental design and treatments:

A randomized complete block design was used with fourteen treatments, including control. It consisted of five replicated pots and one plant per pot. Pepper seedlings were treated with extract by spraying whole leaves, even run-off, with different (PLE) concentrations after 20 and 40 days as follows:

1-pepper seedling untreated control.

2- Pepper seedlings foliar with 25 % (SPLE) +75 % tap water.

3- Pepper seedlings foliar with 50 % (SPLE) + 50% tap water. 

4- Pepper seedlings foliar with 100% (SPE).  

5- Pepper seedlings foliar with 25 % (APLE) +75 % tap water.

6- Pepper seedlings foliar with 50 % (APLE) +50 % tap water

7- Pepper seedlings foliar with 100 % (APLE).

8- Pepper seedling infected (PMMoV) without any treated.

9- Pepper seedlings foliar infected (PMMoV) treated with 25 % (SPLE) +75 % tap water.

10- Pepper seedlings infected (PMMoV) foliar with 50 % (SPLE) +50 % tap water.

11- Pepper seedlings infected (PMMoV) foliar with 100 % (SPLE).

12- Pepper seedlings foliar infected (PMMoV) with 25 % (APLE) +75 % tap water.

13- Pepper seedlings infected (PMMoV) foliar with 50 % (APLE) +50 % tap water. 

14- Pepper seedlings infected (PMMoV) foliar with 100 % (APLE). 

Gas chromatography (GC) analysis:

Varian 3400 chromatograph. line, 30 cm in height and 0.32 mm in width, was working with helium as a transporter gas. GC temperature software program. Spectra of mass were saved in electron ionization (EI) form at 70 eV. The check repetition ranged over a mass of atomic mass units.

Statistical design and analysis:

The design was a completely randomized block (RCBD) with five replicates. For each treatment, The least significant differences (LSD) was used to test the differences among the means of each parameter.

Results

Populus nigra leaf extract chemical composition in autumn and spring:

The investigated data indicated that there were differences in the chemical structure of P. nigra extract between spring and autumn periods. It was clear that spring (PLE) extract more enhanced the chemical composition present compared to the autumn extract period. In comparison to the autumn (PLE) extract, the spring (PLE) extract contained more tiglic acid, phenol, benzoic acid, dihydrocinnamic acid, cinnamic acid, 4-hydroxyphenylacetic acid, 4-methoxycinnamic acid, 3,4-dimethoxymethyl cinnamate, ferulic acid, caffeic acid, and pinostrobin chalcone as shown on Table (2).

Table (2): Chemical composition of P. nigra leaves extract during autumn and spring

Effect of P. nigra spring leaves extract on C. annuum growth:

P. nigra spring leaf extracts significantly suppressed C. annuum growth parameters compared to control. According to the investigated data, using 25% foliar application from (PLE), had the lowest significant decrease in pepper growth parameters. Peppers treated with PLE foliar at various concentrations, on the other hand, had a reduction effect on (PMMoV) infected peppers when compared to untreated infection peppers. The second season had the same trend. According to Table (3).

Table (3): Effect of foliar application of P. nigra spring leaves extract (PLE) and inoculation with pepper mild mottle virus (PMMoV) on morphological characters of pepper plants in the two 2019 and 2020 seasons

Effect of Populus nigra autumn leaves extract on Capsicum annuum growth:

Data on Table (4) recorded that treated C. annuum with different autumn leaves extract concentrations of P. nigra (PLE) had a significant increase in vegetative growth compared to control. In thisregard, the (100 PLE) percentage demonstrated a highly significant increase when compared to the (25 and 50) PLE percentages. When compared to untreated infected pepper, foliar application of (PLE) results in a significant increase in recovering infected C. annuum with (PMMoV).Meanwhile, treating 100 PLE percentage showed the most improvement compared to other concentrations. In general, the second season followed the same pattern.

Table (4): Effect of foliar application of P. nigra autumn leaves extract (PLE) and plants infected with pepper mild mottle virus (PMMoV) on morphological characters of pepper plants in the two 2019 and 2020 seasons

Effect of (PLE) after 20 and 40 days from (PMMoV) on symptoms:

Using (SPLE) foliar application with different concentrations, processed infected pepper plants enhanced fruit pepper virus symptoms compared to untreated infected fruit peppers. As regard to Table (5).

Table (5):Effect of foliar application of P. nigra spring leaves extract (SPLE) after 20 and 40 days from PMMoV inoculation on external symptoms % of pepper plants in the two seasons (2019 and 2020)

Tab.(6):Effect of foliar application of autumn Populus nigra leaves extract (APLE) after after 20 and 40 days from PMMV inoculation on external symptoms of pepper plants in the two seasons (2019 and 2020)

Data in Table (7) indicated that treated infected pepper with spring (PLE) foliar application enhanced pepper defense for (PMMoV) compared to untreated. By using (PLE100) recorded the highest significant decrease in treatment after the first and second. The second season had the same trend. On the other hand, autumn (PLE) applications lead to significantly decreased virus concentration and reproductive.

Table (7): Effect of foliar application of P. nigra leaves extract (PLE) after first and second from PMMoV inoculation on relative concentration of PMMoV with ELISA of pepper plants in the two seasons (2019 and 2020).

Discussion

Leaves of Populus sp turn yellow in autumn as a consequence of chlorophyll degradation during senescence in response to environmental change. Hoch, et al., (2003) and Landi, et al., (2015). The present study indicated that concentrations of phenolic acids in leaves were highly elevated at the beginning of the spring period and decomposed at the end of the active season until leaves maturity Riipi, et al., (2002). Barros, and Neill, (1987) indicated that willow trees' degraded structure and growth rate turn down on senescence leaves in relation to low leaf water potential. Senescence process leaf damage caused by uncoupled chlorophyll that downstream decreased led to the photosynthesis process not operating and increased chemical decomposition in Hörtensteiner, (2006); Hörtensteiner, (2009). Moreover, 75.0% diluted ethanol solvent had a significant effect in extracting phenol composition. Sun, et al., (2015).

In fact, chlorophyll loss might be a sign of membrane damage, especially if hydrogen peroxide generation is enhanced Nishiyama, et al., (2011). By destroying a variety of targets, including proteins, reactive oxygen species can cause significant harm to cell structure and metabolism Hussain, et al., (2017). In fact, chlorophyll loss might be a sign of membrane damage, especially if hydrogen peroxide generation is enhanced Nishiyama, et al., (2011). Reactive oxygen species have the potential to cause significant cell damage. Indeed, phenolic compounds appeared to reduce seedling growth of crop. Blum, et al., (1991). Meanwhile, phenol structures caught hold of the behaviour of respiratory enzymes. Typically pretentious phenol components are aldolase plus glucosephosphate isomerase, involved in glycolysis and glucose 6-phosphate dehydrogenase. Muscolo, et al., (2001).

On the other hand, Populus sp. is a genus of the Fam. Salicaceae, and includes different naturally-happening aromatic components such as salicylic acid and salicylic alcohol, as well as aromatic ketones moreover terpenoids furthermore fatty addition to organic acids plus benzyl alcohol also beta-phenylethanol, moreover other compounds. Pearl, and Darling, (1970); Steele, and Ronald, (1973). The researchers also indicated that the majority of these structures showed antimicrobial motion, such as salicylic component, Bartzatt, et al., (2007).

In the study present that aspen had different concentrations of deterrent secondary substances relating to bud grow old. This concerning for herbivores had incapable feed selective as defense protection. Mattes, et al., (1987); Reichardt, et al., (1990) and Clausen, et al., (1992). Poplar trees had surprising occurrence famous as autumn senescence a vital role to survival trees Ghelardini, et al., (2014). The commencement of inception senescence had known altering in metabolic rate of leaf since starting copious photosynthetically active to senescing situation then leaf actively had been incapable precious components content after that transported out leaf Fischer, and Feller, (1994). Senescence leaves is a greatly synchronized development in cell organs. It had deconstructed and content reallocated Woo, et al., (2013). While the tree cell get the beginning pointer for preparatory programmed death. Cell started to dismantle itself then manner initial chlorophyll molecules after that nucleus and mitochondria dismantled Van Doorn, and Woltering, (2004).

Fariduddin, et al., (2003) indicated that the exogenous function of salicylic acid enhanced the photosynthetic. Meanwhile, Shakirova, et al., (2003) observed the encouraging consequence of low salicylic acid concentration on increased yield. When cucumber and tomato plants were treated with reduced salicylic acid concentrations, fruit output parameters enhanced considerably Larque-Saavedra, and Martin-Mex, (2007). On this concern Yildirim, and Dursun, (2009) found that foliar application of salicylic acid had a positive effect on early yield and total yield, and that the highest yield occurred in the 0.50 mM salicylic acid treatment. They also suggested that in order to improve yield, foliar application of salicylic acid be used. On this mention low doses, salicylic acid more capable photosynthesis and growth parameters than excessive amounts Kurepin, et al., (2013).

Higher salicylic acid concentrations [10^− 4 M] inhibited ethylene synthesis, according to Srivastava, and Dwivedi, (2000). However, the mechanism of action of salicylic acid-mediated ethylene biosynthesis is still unknown. As a result, elucidating and pinpointing the mechanism connected with salicylic acid for ethylene biosynthesis and action will require a lot of debate. On this side, Pancheva, et al., (1996) found chlorophyll reduced following concentrations (100 mM to 1 mM) of salicylic acid submission in leaves. Boatwright, et al., (2013) reported that some virus-encoded proteins have demonstrated the ability to inhibit or enhance salicylic acid dependent signaling.

Salicylic acids enhance the confrontation mechanisms of phytoalexin construction, in addition to being capable of cell wall membrane amplification and lignification. Furthermore, salicylic acid submission on tobacco improved the confrontation with viruses and the resistance movement among plant cells. Mayers, et al., (2005). Raskin, (1992) investigated that salicylic acid, an important component, encourages endogenous messengers which enhance pathogen resistance. This theory has maintained tobacco plants' foliar application by salicylic acid, and this improvement persuaded structures of pathogenesis related proteins as well as confrontation with tobacco mosaic virus. Van Huijsduijnen, et al., (1986). In this admiration, Zhao, et al., (2017) reported that, instruction of virus resistance with phenol stricture is possible. Hackmann, et al., (2014) found that salicylic acid phytohormone participates in modifiable protection mostly against biotrophic moreover hemibiotrophic pathogens.

The preceding study indicated that salicylic acid reduced virus concentrations detected by DAS-ELISA. The diminution in virus concentrations released an enhanced peroxidase enzyme, which is recognized to encourage forming polymerization. It lead to lignin combination plus point had straight connected with augmented facility of systemically secluded lignin tissues furthermore assist protection from viral infection. Chittoor, et al., (1999). On this concern salicylic acid excesses production of antioxidants moreover improved virus resistance. Furthermore, accumulation of salicylic acid following the process could be radically induced in plants challenged for various viruses. Zhao, et al., (2019). An improvement in salicylic acid concentration was essential for plants' resistance to viral infection and virus replication Zhao, et al., (2019).

Conclusion

Pepper is economic and popular crop. In this study, virus damaged pepper crops without any infect side. According to this study recommended treated with different concentrations of (APLE) and (SPLE) foliar application an able to inhibitor virus activity. On other side using (APLE) significant enhance pepper fruit production.

Declarations

Ethics approval and consent to participate: applicable 

Consent for publication: yes, we agree for publication.

Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests: The author declares that he has no competing interests.

Funding: Not applicable. 

Author’s contributions 

The authors equally working on data collection, data analysis and interpretation, and manuscript writing. The author read and approved the final manuscript. 

All the plant experiments were in compliance with relevant institutional, national, and international guidelines and legislation.

Acknowledgement: : The authors gratefully acknowledge Prof. Dr. Ahmed M. Soliman (Virus & Phytoplasma Res. Dept., Plant Pathol. Res. Institute, Agricultural Research Center, Egypt) for his valuable assistance during the preparation of this manuscript.

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