Ecofriendly Bioagents, Parthenocissus quinquefolia , and Plectranthus neochilus Extracts to Control the Early Blight Pathogen ( Alternaria solani ) in Tomato

: Background: early blight disease caused by Alternaria solani is one of the most destructive diseases of the tomato, reducing tomato production globally. Methods: four fungal isolates were collected from four tomato cultivars and identified through morphological characterization and polymerase chain reaction (PCR) amplification of the internal transcript spacer (ITS) region. Plectranthus neochilus and Parthenocissus quinquefolia methanol extracts and the bioagents Trichoderma viride and Pseudomonas ﬂuorescens were used as antifungal agents in vitro and in vivo and compared with chlorothalonil, a reference chemical fungicide. HPLC analysis of the plant extracts was used to identify the main ﬂavonoid compounds, namely, rutin and myricetin. Results: molecular characterization showed that the fungal isolates belonged to A. solani . The results of in vitro antifungal activity studies revealed that chlorothalonil, at a concentration of 2500 mg/L, showed the highest inhibition percentage of fungal growth (IPFG) against A. solani (84.4%), followed by the bioagents T. viride and P. ﬂuorescens , with IPFG values of 72.9% and 67.9%, respectively. Moderate to weak activity was found against A. solani when P. neochilus and P. quinquefolia extracts were applied at a concentration of 2500 mg/L, with an IPFG value of 54% for both extracts. The results of in vivo spray application showed that T. viride and chlorothalonil, as well as P. ﬂuorescens , signiﬁcantly reduced the disease index of early blight, and followed by the P. neochilus and P. quinquefolia extracts. By HPLC, the ﬂavonoid compounds rutin and myricetin were identiﬁed in P. neochilus (leaf) with amounts of 2429.60 and 75.92 mg/100 g of extract, and in P. quinquefolia (fruit), with amounts of 1891.60 and 241.06 mg/100 g of extract, respectively. Conclusions: the results of the bioactivity of plant extracts and the bioagents indicate a vital role as antifungal activity against A. solani .


Introduction
The tomato (Solanum lycopersicum L.) is one of the most important vegetable crops [1]. It is susceptible to various diseases caused by different pathogens, such as bacteria, viruses, nematodes, and fungi [2]. Early blight disease, one of the most common tomato diseases, is Pure cultures of the fungal pathogen were obtained by the single-spore isolation method, and these cultures were used for pathogenicity tests by following Koch's postulates [43]. Healthy tomato fruit were taken, and their surfaces were sterilized with ethanol (70%). Artificial infection was carried out using 5 µL of the fungal spore suspension; a spore suspension with a concentration of 3 × 10 6 spore/mL was used to inoculate each tomato, where the suspension was placed on each fruit, and the fruit was placed under humidified conditions in an incubator at 27 • C for one week [44].

Tomato Seedlings
Seeds of the tomato cultivar Dosera were grown in a greenhouse, and the soil used for cultivation was sterilized by an autoclave. The temperature for plant growth was maintained at 28 • C to 32 • C, and the relative humidity was maintained at 40 to 60%; the plants were allowed to attain a height of 150-200 mm. The collection of tomato plants have been done under the permission at Agriculture Research Center (ARC), Alexandria, Egypt.
Four replications were used in the pathogenicity test. A spore suspension with a concentration of 3 × 10 6 spore/mL (containing 0.01% Tween 20) was sprayed on leaves, and the degree of leaf infection was studied by visual observation of the extent of lesion development on the leaves, which was assessed for 10 days after inoculation [45]. Observations of the severity of the disease on the foliage were recorded using a 0-5 scale, as shown in Table 1 [46], and the percentage disease index (PDI) was determined by using the following formula and the description of the disease scale [47]: PDI = (A/B × C) × 100, where A is the sum of all ratings, B is the number of plants, and C is the maximum rating.

. Cultural and Morphological Characteristics
The fungal isolates were identified by microscopic examination, including examination of the structure, size, and shape of the conidia. The isolates were identified according to the criterial for cultural and morphological characteristics described by Naik et al. [41].

Molecular Characterization via Polymerase Chain Reaction (PCR) Amplification of the Internal Transcript Spacer (ITS) Region
After obtaining pure cultures of the fungal isolates, DNA was extracted from these isolates using a rapid mini-preparation procedure [48,49]. The ITS DNA region of these isolates was amplified via PCR using the universal primers ITS1 (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS4 (5 -TCCTCCGCTTATTGA TATGC-3 ), which amplified the ITS regions and 5.8S genes encoded by fungal species. Amplification of the ITS rDNA was performed in a total volume of 25 µL, containing 12.5 µL of PCR Green Master Mix (Thermo Scientific™, Gloucester, United Kingdom), 3 µL of template DNA, 8.5 µL of molecular-grade water, and 0.5 µL each of the universal forward primer (ITS1) and reverse primer (ITS4). The optimized thermal profile for PCR was as follows: initial denaturation at 95 • C for 3 min; 35 cycles of denaturation at 94 • C for 30 s, and annealing at 55 • C for 2 min; and a final extension at 72 • C for 10 min. The PCR products were separated on a 1.5% agarose gel in 0.5X Tris-borate-EDTA (TBE) buffer at 65 volts for 15 min, run parallel to a standard DNA molecular marker, and visualized under a UV transilluminator.

Sequencing of the ITS Region and Phylogenetic Analysis
The obtained ITS rDNA (500-700 bp) regions of selected isolates were sent for sequencing (Macrogen, Scientific Services Company, Seoul, Korea). The sequences were compared to those in GenBank (http://www.ncbi.nlm.nih.gov, accessed on 4 April 2020) using NCBI BLAST. The sequences obtained were submitted to GenBank. The ITS sequences of fungal strains were downloaded from the GenBank database and used in the phylogenetic analyses as reference sequences. All the DNA sequences were aligned with the program CLUSTALW [50]. The resulting multiple-alignment file was used for phylogenetic analyses. The evolutionary history was inferred using the Maximum Parsimony method. The evolutionary history was inferred using the Maximum Parsimony method. The consensus tree inferred from 10 most parsimonious trees is shown. Branches corresponding to partitions reproduced in less than 50% trees are collapsed. The consistency index is 1.000000 (1.000000), the retention index is 1.000000 (1.000000), and the composite index is 1.000000 (1.000000) for all sites and parsimony-informative sites (in parentheses). The percentage of parsimonious trees in which the associated taxa clustered together are shown next to the branches. The MP tree was obtained using the subtree-pruning-regrafting (SPR) algorithm [51] with search level 0, in which the initial trees were obtained by the random addition of sequences (10 replicates). The analysis involved 10 nucleotide sequences. Sequence gaps were treated as missing data. There were a total of 288 positions in the final dataset. Two biological control agents, namely, Trichoderma viride (accession no. MW647090) and Pseudomonas fluorescens (accession no. MW647093), were evaluated for their efficacy against the fungal pathogen using a dual-culture technique [19,49]. Fifteen milliliters of PDA was poured into 9-cm-diameter Petri dishes and allowed to solidify. A 0.5-cm disc of the pathogen was taken from growing margins of a 7-day-old culture and placed at one end of the Petri dish. The T. viride strain isolated in this study from an infected tomato field (0.5 cm disc) was inoculated on the opposite side of the same Petri dish. In the case of the bacterial antagonist, the fungus was centered between two P. fluorescens lines in Petri dishes and incubated for 7 days at 27 • C. The activity of the antagonistic organisms was recorded by measuring the colony diameter in each treatment and comparing it to the control value [52].

Plant Extracts and Their Bioactivity In Vitro
P. quinquefolia fruit and P. neochilus leaves were collected from Alexandria, Egypt. The samples were air-dried under laboratory conditions and ground using a small laboratory Wiley mill. Approximately 50 g each of the P. quinquefolia fruit and P. neochilus leaf samples were extracted with 200 mL of methanol for three days at room temperature and then filtered using Whatman No. 1 filter paper [53]. Subsequently, the solvent was evaporated, and the extracts were concentrated under vacuum using a rotary evaporator at 45 • C. Furthermore, the crude extracts were stored in sealed vials at 4 • C until further use for in vitro screening of antimicrobial activity [54].
The extracts were prepared at concentrations of 2500, 1250, and 625 mg/L by dissolving the extract in dimethyl sulfoxide (DMSO 99.99%) and tested against the growth of the isolated fungus. Wells with a 6-mm diameter were cut out from the PDA medium and filled with 80 µL of each extract. Fungal isolates were grown on PDA and placed at one end of the Petri dish. Each antimicrobial assay was performed in triplicate. The plates were incubated at an appropriate growth temperature (27 • C) for 7 days. The assessment of antimicrobial activity was based on the measurement of linear growth of fungi on the agar surface around the well [55].

In Vitro Evaluation of Fungicide
The efficacy of the fungicide chlorothalonil (Brado 72% SC ® ) as a chemical positive control was tested against the isolated fungus at three concentrations: 2500 (1×) Ministry of Agriculture Recommendation), 1250, and 625 mg/L. The fungicide was added to PDA medium after sterilization. A 0.5-cm disc of the fungal isolate was removed and placed at the center of a Petri dish and incubated for 7 days at 27 • C, and the activity of the fungicide was recorded by measuring the colony diameter of the tested fungus in each treatment and comparing it to the control value [58,59].
The bioagents, plant extracts, and chemical positive control (chlorothalonil) were tested with a completely randomized design in triplicate. Then, the plates were incubated until fungal growth covered the surface of PDA medium in the control treatment [60]. The efficacy of each treatment was determined by measuring linear growth (cm), and the data are expressed as the percentage of mycelial growth inhibition compared with the control using the following formula [19,61]: mycelial growth inhibition (%) = (T 0 − T a /T 0 ) × 100; where T 0 and T a are the average diameters (mm) of fungal colonies under the control and experimental treatments, respectively.

Control of Early Blight Disease in Tomato In Vivo
The biocontrol agents and plant extracts as well as the chemical fungicide were screened on tomato seedlings. After the plants attained a height of 20 cm, a spore suspension of the fungal isolate (A.s.1) containing 3 × 10 6 spore/mL was sprayed on the tomato seedlings. T. viride (10 6 spore/mL), and P. fluorescens (10 8 CFU/mL), as well as the plant extracts and fungicide (2500 mg/L), were sprayed onto the tomato seedlings one day after inoculation. The experiment was conducted in a randomized complete block design with four replications. Data on disease severity were obtained after three weeks of all treatments [62]. The percent disease index (PDI) was calculated [63], as was the percent reduction in PDI (%) [2].

Statistical Analysis
The reduction in linear growth of the pathogen as an effect of treatment with biotic and biocontrol agents was analyzed using analysis of variance in a completely randomized design using a computer program, Statistical Analysis System (SAS), and compared with the values for of the control. Means among the treatments were compared using minimum significant difference measured by Tukey's Studentized Range (HSD) Test at Alpha 0.05 [64].

Isolation of the Fungal Pathogen
Four fungal isolates of the early blight disease pathogen (Alternaria spp.) were obtained from field-infected plants of the tomato cultivars Dosera, 023, Marina Hajin F2, and Ajyad7.

Tomato Fruits
The pathogenicity test of the four isolates showed their ability to infect artificially inoculated tomato fruits after one week, in which black spots appeared around the infected area, and fungal growth increased as the incubation period increased (Figure 1). and biocontrol agents was analyzed using analysis of variance in a completely randomized design using a computer program, Statistical Analysis System (SAS), and compared with the values for of the control. Means among the treatments were compared using minimum significant difference measured by Tukey's Studentized Range (HSD) Test at Alpha 0.05 [64].

Isolation of the Fungal Pathogen
Four fungal isolates of the early blight disease pathogen (Alternaria spp.) were obtained from field-infected plants of the tomato cultivars Dosera, 023, Marina Hajin F2, and Ajyad7.

Tomato Fruits
The pathogenicity test of the four isolates showed their ability to infect artificially inoculated tomato fruits after one week, in which black spots appeared around the infected area, and fungal growth increased as the incubation period increased (Figure 1).

Tomato Seedlings
Artificial infection of tomato seedlings was carried out under greenhouse conditions on 15-day-old seedlings of the tomato cultivar Dosera. The disease symptoms were observed one week after inoculation as browning of the tissue followed by necrosis. The spots produced were oval in shape, and in the later stage, these spots expanded, and concentric circles were formed and were surrounded by a yellow halo. Finally, the spots changed from brown to dark brown (Figure 2), and the non-inoculated plants did not exhibit disease symptoms. Table 2 shows the percent disease index (PDI), and the degree of leaf infection was investigated by visual observation.

Tomato Seedlings
Artificial infection of tomato seedlings was carried out under greenhouse conditions on 15-day-old seedlings of the tomato cultivar Dosera. The disease symptoms were observed one week after inoculation as browning of the tissue followed by necrosis. The spots produced were oval in shape, and in the later stage, these spots expanded, and concentric circles were formed and were surrounded by a yellow halo. Finally, the spots changed from brown to dark brown (Figure 2), and the non-inoculated plants did not exhibit disease symptoms. Table 2 shows the percent disease index (PDI), and the degree of leaf infection was investigated by visual observation.

Cultural and Morphological Characteristics
Four purified fungal isolates, namely, A.s.1, A.s.2, A.s.3, and A.s.4, were identified based on morphological characteristics. The conidia were brown to olivaceous brown, were solitary and straight or exhibited ellipsoidal tapering, and had transverse and longitudinal septate. According to the microscopic images of the four pathogenic fungi and preliminary evaluation, all the isolates belonged to A. solani. The color of the observed colonies was dark brown or olivaceous brown, and the colonies were smooth on PDA medium (Figure 3).

Molecular Characterization through Sequence Analysis of the ITS Region and Phylogenetic Tree of Alternaria Solani Isolates
The fungal isolates were identified via amplification and sequencing of the ITS region. The four isolates produced a PCR product of approximately 500-700 bp, and the ITS sequences were submitted to GenBank. The accession numbers are listed in Table 2. The accession numbers of the ITS sequences were MT279570, MT279571, MT279572, and MT279573. The DNA sequence obtained for each fungal isolate showed 99.5% homology with the A. solani sequences available in GenBank, as determined by utilizing the BLAST tool. The level of similarity reported here confirms the morphological identification of the isolates. In the phylogenetic tree of the ITS region, four A. solani isolates obtained in this study were compared with six isolates collected from GenBank (GU395512, MT135014,

Molecular Characterization through Sequence Analysis of the ITS Region and Phylogenetic Tree of Alternaria Solani Isolates
The fungal isolates were identified via amplification and sequencing of the ITS region. The four isolates produced a PCR product of approximately 500-700 bp, and the ITS sequences were submitted to GenBank. The accession numbers are listed in Table 2. The accession numbers of the ITS sequences were MT279570, MT279571, MT279572, and MT279573. The DNA sequence obtained for each fungal isolate showed 99.5% homology with the A. solani sequences available in GenBank, as determined by utilizing the BLAST tool. The level of similarity reported here confirms the morphological identification of the isolates. In the phylogenetic tree of the ITS region, four A. solani isolates obtained in this study were compared with six isolates collected from GenBank (GU395512, MT135014, KX452728, KF999007, HQ270456, and MT199327), and high genetic similarity to the reported isolates was found (Figure 4).

Evaluation of Bioagents, Plant Extracts and a Chemical Fungicide against A. solani Isolates In Vitro
The data presented in Table 3 show the highly significant effects of the tested biocontrol agents T. viride (Accession no. MW647090) and P. fluorescens (accession no. MW647093) and plant extracts (P. neochilus and P. quinquefolia) against the growth of A. solani isolates compared with the effect of chlorothalonil 72% as a chemical positive control.

Evaluation of Bioagents, Plant Extracts and a Chemical Fungicide against A. solani Isolates In Vitro
The data presented in Table 3 show the highly significant effects of the tested biocontrol agents T. viride (Accession no. MW647090) and P. fluorescens (accession no. MW647093) and plant extracts (P. neochilus and P. quinquefolia) against the growth of A. solani isolates compared with the effect of chlorothalonil 72% as a chemical positive control. It is evident that chlorothalonil 72% was the most suppressive agent against all the A. solani isolates, with inhibition percentage of fungal growth (IPFG) values that ranged from 84.44% against the isolate A.s.3 at a concentration 2500 mg/L to 77.78% against the isolate A.s.1 at the same concentration. In addition to the positive chemical control (chlorothalonil), the biocontrol agents T. viride and P. fluorescens were found to have high IPFG values of 72.99% and 67.93%, respectively. Moderate to weak activity was found against the A. solani isolate when the P. neochilus and P. quinquefolia extracts were applied at a concentration of 2500 mg/L, and the highest IPFG value (54.01%) of both extracts was observed against the A. solani isolate (A.s.1). The lowest IPFG value (31%) of both extracts was observed against the A.s.3 isolate at the same concentration (2500 mg/L) ( Figure 5). Moreover, as shown in Table 3, as the concentrations of chlorothalonil and the P. neochilus and P. quinquefolia extracts increased, the IPFG values against the growth of A. solani isolates increased.

HPLC Analysis of Flavonoids in P. neochilus and P. quinquefolia Extracts
The main flavonoid compounds identified in the P. neochilus (leaf) and P. quinquefolia (fruit) methanolic extracts by HPLC analysis were rutin (2429.60 and 1891.60 mg/100 g of plant extract, respectively) and myricetin (75.92 and 241.06 mg/100 g of plant extract, respectively) ( Table 4). The HPLC chromatograms of the flavonoids identified in the P. neochilus and P. quinquefolia extracts are summarized in Figure 6a,b.      Table 5 shows the efficacy of the bioagents T. viride and P. fluorescens, as well as the P. neochilus and P. quinquefolia plant extracts and the fungicide chlorothalonil, in reducing the severity of early blight disease in vivo. The data were recorded after 21 days of application. Table 5 shows that all the bioagents and plant extracts tested, in addition to the fungicide chlorothalonil, reduced, though to different extents, the disease index of A. solani compared to that of the inoculated control. It was evident that T. viride and chlorothalonil were superior to all the other treatments in reducing the disease severity (80%) of A. solani, followed by P. fluorescens (70%). The plant extracts of P. neochilus and P. quinquefolia showed moderate effects on the reduction in the disease index (70 and 65%), respectively, compared to the control (Table 5).

Evaluation of Bioagents, Plant Extracts and a Chemical Fungicide against A. solani Isolates
In Vivo Table 5 shows the efficacy of the bioagents T. viride and P. fluorescens, as well as the P. neochilus and P. quinquefolia plant extracts and the fungicide chlorothalonil, in reducing the severity of early blight disease in vivo. The data were recorded after 21 days of application. Table 5 shows that all the bioagents and plant extracts tested, in addition to the fungicide chlorothalonil, reduced, though to different extents, the disease index of A. solani compared to that of the inoculated control. It was evident that T. viride and chlorothalonil were superior to all the other treatments in reducing the disease severity (80%) of A. solani, followed by P. fluorescens (70%). The plant extracts of P. neochilus and P. quinquefolia showed moderate effects on the reduction in the disease index (70 and 65%), respectively, compared to the control (Table 5).

Discussion
Early blight disease in tomato caused by Alternaria species is known as a severe and destructive fungal disease in Egypt [65]. Four isolates of the fungus A. solani, which is associated with early blight in tomato plants, were investigated. These isolates were obtained from different tomato fields in Egypt. Morphological identification of the fungal isolates was performed according to the morphological characteristics reported by Simmons, such as colony morphology, size, and shape of conidia and pattern of conidial septation among the tested isolates [66]. The characteristics of this pathogen were consistent with the characteristics described by the Commonwealth Mycological Institute, Kew, Surrey, England [67]. Thus, the pathogen was identified as A. solani [68], according to morphological characterization and amplification and sequencing of the ITS region. Based on previous studies, A. solani is the main cause of early blight disease in the family Solanaceae [69]. Furthermore, molecular techniques are suitable methods for analysis, particularly for researchers who are not familiar with the conventional characterization of fungi [70]. One of these methods is sequencing of the ITS region of ribosomal DNA, which distinguishes Alternaria spp. from other pathogens very well [71,72]. The data obtained from molecular studies confirmed the morphological characterization of the tested fungal isolates obtained in this research [7,73,74].
Recently, there has been increasing concern regarding the use of ecofriendly bioagents to control plant pathogens [19,75,76]. Moreover, biological control of early blight pathogens is an attractive alternative to conventional chemical control through the selection and exploitation of fungal and bacterial strains antagonistic to the pathogens that cause early blight in tomato [77][78][79]. The genus Pseudomonas contains a number of strains that are useful for plant protection [80], for example, strains with the ability to produce antibiotics and siderophores [81]. The results obtained from in vitro and in vivo studies revealed that T. viride and P. fluorescens isolates were suppressive to A. solani isolates, and disease severity was investigated [82][83][84]. Additionally, these results are consistent with those of Casida and Lukezie, who reported that Pseudomonas strain 679-2 was able to reduce the severity of early blight disease caused by A. solani [85].
Furthermore, Trichoderma viride, due to its antagonistic activity, is considered a potential biological control agent against many plant pathogenic fungi [49,86]. Trichoderma sp. controls pathogen growth via the production of extracellular enzymes, antibiotics, and antifungal metabolites [16,84,87,88]. The results of this study are consistent with the observed effectiveness of four fungicides, chlorothalonil, copper chloride oxide, and azoxystrobin, at different concentrations against A. solani; the results showed that the fungicides significantly reduced the radial growth of the tested isolates of A. solani [89,90].
Plant defense responses involve the activation of multiple coordinated and apparently complementary defense reactions involving the production of phytoalexins or other antimicrobial compounds, the formation of physical barriers through increased cross-linking, and elicitation of the hypersensitive response [91][92][93]. The induction of plant defense responses also seems to involve several signal transduction cascades [94].
Many attempts have been made to biologically control plant diseases through induction of resistance in the host against the corresponding pathogen by saprophytic bacte-  [95], including the use of Pseudomonas fluorescens to control of tobacco mosaic virus [96] and in rice against sheath blight disease [97].
A number of biochemical alterations observed following treatment with bacterial inducers of systemic resistance, such as induction of phytoalexins; induction and/or stimulation of key enzymes, including peroxidase, phenylalanine ammonia lyase, chalcone synthase, chitinase, and β-1,3-glucanase [98][99][100]; and stress-related proteins, have been implicated in the mechanism of resistance [100]. The accumulation of phenolics, callose deposition, and lignification have also been reported and linked to the phenomenon of acquired resistance [101][102][103].
The P. neochilus and P. quinquefolia extracts showed moderate effects against the early blight pathogen A. solani in vitro and in vivo. These results are in line with those of El-Hefny et al. [56], who used an acetone extract of Withania somnifera fruit, which contains flavonoids (rutin and myricetin) at 3%, to inhibit the growth of fungal mycelia of Fusarium culmorum by 84.07% and Rhizoctonia solani by 67.03%.
Flavonoids have been proposed to control fungal pathogens via their inhibitory effect on fungal spore germination [104,105]. Moreover, they cause disruption of the fungal plasma membrane, induction of mitochondrial dysfunction and inhibition of cell wall formation, cell division, RNA, and protein synthesis and the efflux-mediated pumping system [106]. Furthermore, flavonoid compounds, such as catechin and rutin extracted from pomegranate peel, have potent inhibitory effects against Colletotrichum gloeosporioides, a fungus that infects Persea americana [107]. Flavonoids inhibit many varieties of eukaryotic enzymes, and this inhibition of enzymes may be due to the interaction of enzymes with different parts of the flavonoid molecule, such as carbohydrates, phenyl rings, phenols, and benzopyrone rings [108]. Moreover, the antimicrobial activity of P. neochilus extracts has been associated with the lipophilicity of their chemical constituents, mainly monoterpenes and sesquiterpenes, which are often the main chemicals therein [23,109]. The antimicrobial activity of P. quinquefolia may be due to some phenolic compounds in these extracts [110].

Conclusions
In agriculture, there is an important need for alternate ecofriendly materials to control plant diseases. This study provides insights for the development of new phytosanitary products based on plant extracts of Plectranthus neochilus and Parthenocissus quinquefolia and on the bioagents Trichoderma viride and Pseudomonas fluorescens for the control of A. solani in tomato plants. The tested bioagents, plant extracts, and the fungicide chlorothalonil were significantly reduced disease index of A. solani. T. viride and chlorothalonil were suggested to be superior in the reduction of disease severity of A. solani followed by P. fluorescens extract. In vivo, and with spray application of the tested agents, T. viride, chlorothalonil followed by P. fluorescens extract were observed the most reduction in the disease index of early blight. This study suggested and recommended alternatives to chemical pesticides to achieve organic production.