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.
Family
|
Host plant
|
Symptoms
|
Chenopodiaceae
|
Ch. amaranticolor Coste and Reyn
Ch. quinoa Wild
Beta vulgaris
|
CLL
CLL NS
|
Cucurbitaceae
|
Cucurbita pepo cv. Cavili
Cucurbita pepo cv. Eskandarni
Cu. maxima cv. Wintersquash
Cucumis sativus cv. Balady
Citrullus lanatus cv. Giza 2
|
NS
NS
NS
NS NS
|
Fabaceae
|
Glycine max L.cv.Giza22
Lupinus termis cv. Lupine
Phaseolus vulgaris cv. Giza 4
Pisium sativum L. cv. Sugar sweet
Vicia faba cv. Giza 3
|
NS
NS
NS
NS NS
|
Solanaceae
|
Capsicum annum L. cv. California
Capsicum fratescens L.cv. Chilli
Datura metal
Datura stramonium
Nicotiana tabacum L.cv.Whit Burley
|
M+Y
M+Mf
NLL
NLL
NLL
|
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.