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Plants from a semi-arid environment as a source of phytochemicals against Fusarium crown and foot rot in zucchini

Plants from a semi-arid environment as a source of phytochemicals against Fusarium crown and foot... Fusarium crown and foot rot, caused by F. solani f. sp. cucurbitae, are major fungal diseases affecting zucchini and other cucurbits. Despite the efficacy of synthetic fungicides, their health and environmental hazards have highlighted the urgent need for safer alternatives, such as phytochemical‑based biocides. Owing to the upregulation of the plant sec‑ ondary metabolism under stressful conditions, bioprospecting in harsh environments could reveal ore plants for bio‑ active metabolites. In this study, thirteen wild plants were collected from their natural habitat in a semiarid environ‑ ment (Yanbu, Saudi Arabia) and extracted to obtain phenolics rich extracts. Total polyphenols, flavonoids, antioxidant capacities and the antifungal activities of the extracts against a pathogenic isolate of F. solani were assessed. Fusarium solani was isolated from infected zucchini and characterized by scanning electron microscopy. Hierarchical clustering analysis of the phytochemical screening and in vitro bioactivity revealed that Rosmarinus officinalis, Pulicaria crispa, Achillea falcata and Haloxylon salicornicum were the richest in polyphenols and the most powerful against F. solani. Further, the extracts of these four plants significantly decreased the disease incidence in zucchini, where P. crispa was the premier. Interestingly, results of transmission electron microscopy revealed that extract of P. crispa, as a representa‑ tive of the powerful group, induced ultrastructural disorders in fungal cells. Therefore, this study suggests the use of R. officinalis, P. crispa, A. falcata and H. salicornicum grown in semi‑arid environments as ore plants to develop phyto ‑ chemical‑based biocides against Fusarium crown and foot rot. Keywords Phytochemicals, Polyphenols, Biocides, Fusarium solani, Zucchini, Ultrastructure a broad range of host plants (Barreto et  al. 2003), and Introduction attacks the postharvest crops (Akrami et  al. 2012). The Fusarium species are the most common soil-born fungi; diseases caused by F. solani are characterized by many despite being saprophytes, they are well-known as plant symptoms such as root and stem rotting, wilting, leaf yel- pathogens (Coleman 2016). Fusarium solani is a cosmo- lowing, and sudden death (Nemec et al. 1976). Although politan aggressive plant pathogenic fungus that invades the persistence of F. solani in the agricultural soil after disease occurrence is poorly investigated, some formae speciales, such as F. solani f. sp. cucurbitae and fragariae, Ahmed M. A. Khalil and Ahmed M. Saleh have contributed equally to this manuscript and share first authorship have been reported to form chlamydospores that confer survival of phytopathogenic soil borne population after *Correspondence: Mahmoud S. M. Mohamed tillage (Henry et al. 2019). msaleh@sci.cu.edu.eg Many researchers demonstrated that F. solani f. Botany and Microbiology Department, Faculty of Science, Al‑Azhar sp. cucurbitae has host specificity for Cucurbitaceae University, Cairo, Egypt Department of Botany and Microbiology, Faculty of Science, Cairo (Boughalleb et  al. 2007; Paternotte 1987). This phy - University, Giza 12613, Egypt topathogen has been recorded causing disease in field © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Khalil et al. AMB Express (2023) 13:6 Page 2 of 12 production in pumpkin, muskmelon, cucumber, water- compounds have been suggested to attend as valuable melon, and rootstock hybrids in addition to be the causal substitutes to the chemical control of plant pathogens in agent of crown rot for several cucurbits including Zuc- agricultural soils (Langcake 1981). chini squash (Pérez-Hernández et  al. 2017). Plant resist- From a physiological point of view, the production of ance to this pathogen stills limited, while the most biologically active phytochemicals has been reported to common control procedures for the disease caused by be upregulated under adverse environmental conditions F. solani f. sp. cucurbitae are the rotation with non-host (Oh et al. 2009). In this context, due to their severe habi- crops, preventive and curative fungicide application. tat characterized by little water and high temperature, From the human health point of view, application of fun- xerophytic plants have been reported to hyperaccumu- gicides is undesirable as low concentrations of synthetic late phenolic compounds, rendering them a promising pesticides in agricultural products might cause adverse source for antifungal phytochemicals (Fahn and Cutler health effects (Akoto et al. 2013). Further, the accumula - 1992; Yehia et  al. 2020). For instance, Horwoodia dick- tion of pesticides in the agriculture soil may lead to irre- soniae extracts revealed growth inhibitory effect against versible environmental damages such as (i) water-table Aspergillus fumigatus, Streptococcus pneumoniae and and water body contamination, (ii) imbalance in biologi- Escherichia coli (Abdelwahab et  al. 2016). Moreover, the cal diversity, (iii) reduce efficiency of microorganisms antifungal activity of Rhazya stricta extract was per- to degrade pesticides and (iv) develop resistant strains formed against different fungal species including Tricho - (Boughalleb et al. 2007; Abdelkader et al. 2022). phyton longifusis, Aspergillus flavus, Candida albicans Despite the worthwhile results of synthetic fungicides and F. solani. It showed a good inhibitory effect with against F. solani, there is urgent need for developing reducing the hyphal growth of all investigated fungal spe- more safe controlling methods, both for human health cies (Khan and Khan 2007). and environment (Chandel and Deepika 2010; Mostafa Therefore, this study aims to bioprospect in a semi- et  al. 2009). In this regard, the application of plant bio- arid environment (at Yanbu desert, Saudi Arabia) for active molecules and phytochemicals could provide an xerophytes rich in antifungal phenolics, with respect to effectual alternative for synthetic fungicides (Cowan F. solani. Thirteen plants were collected and extracted to 1999; Al Kashgry et  al. 2020). Among these, phenolic obtain phenolics rich extracts. compounds, the most diverse group of plant secondary metabolites including simple phenols, phenolic acids, Materials and methods complex tannins, flavonols and dihydrochalcones, are Isolation and identification of Fusarium solani proved as defensive agents against plant pathogens. Their Fusarium solani was isolated in the laboratory from main advantage is to be environmentally safe as they are infected basal stems of zucchini plants (Cucurbita pepo) easily biodegraded (Steinkellner and Mammerler 2007). displaying external signs of zucchini wilt and crown rot. Several phenolics are reported to accumulate in plant tis- Fusarium solani isolation was achieved by cutting the sues constitutively (i.e. phytoanticipins) and also hyper- infected basal stem into small pieces ranging from 2 to accumulated in response to pathogens challenge (i.e. 3  mm. The pieces were surface disinfected with sodium phytoalexins), playing direct and indirect role in disease hypochlorite (10%) solution for 2 min, washed with ster- resistance (Akhtar and Malik 2000; Lattanzio et al. 2006). ile distilled water several times. Pieces were transferred Generally, antifungal phenolics could act directly by dis- aseptically onto Fusarium selective medium called modi- rupting the fungal morphology, physiology and ultra- fied Nash-Snyder agar (MNSA) (1  g/l KH PO , 0.5  g/l 2 4 structure (Báidez et  al. 2006; Cowan 1999; Khalil et  al. MgSO -7H O, 15  g/l peptone, 20  g/ Agar, 1  g/l penta- 4 2 2020; Nguyen et al. 2013) or indirectly by stimulating the chloronitobenzen, 0.3  g/l streptomycin sulfate, 0.12  g/l plant defense system (Al-Wakeel et al. 2013). The delete - neomycin sulfate) (Nash and Snyder 1962) and incu- rious action of phenolics against fungi could be ascribed bated at 26 ± 2  °C for 5 days. The fungal hyphae were to the presence of acidic hydroxyl group attached to aro- transferred to petri dishes containing potato dextrose matic ring that have lipophilic properties allowing phe- agar medium (PDA). Morphological characteristics were nolic compounds to penetrate the plasma membrane identified both on Czapek-Dox agar (30  g/l sucrose, making ionic homeostatic disturbance (Dambolena et al. 3 g/l NaNO , 0.5  g/l KCl, 100  mg/l F eSO -7H O, 0.5  g/l 3 4 2 2012; Gallucci et al. 2014). Further, these hydroxyl groups MgSO -7H O, 1  g/l K HPO ) and potato dextrose agar 4 2 2 4 could inhibit coupling between the electron transport (PDA) medium as well as microscopic examination and phosphorylation reactions (Parvez et  al. 2004). (Booth 1977; Ellis 1976; Raper and Fennell 1965; Raper Moreover, phenolics have been reported to interfere with and Thom 1949; Rifai 1969) To confirm morphologi - the key enzymes that regulate fungal growth and devel- cal identification, Scanning electron microscopy (SEM) opment (Schwalb and Feucht 1999). Therefore, phenolic was carried out. It was accomplished to approve the K halil et al. AMB Express (2023) 13:6 Page 3 of 12 morphological characteristics of fungal species. A small expressed as mg GAE/ml extract. Total flavonoids con - sample of fungal colony was immersed in glutaralde- tents were assayed by the AlCl colorimetric method hyde (2.5%) for 15 min. The samples were dehydrated by (Sakanaka et  al. 2005). Briefly, 50  µl of each crude plant ethanol-acetone gradient. To avoid collapse in SEM the extract was mixed up to 1.45 ml of distilled water, fol- critical point drying (CPD) was used. Using an Emitech lowed by addition of 75  µl of 5% NaNO solution. The K550X coating unit, the specimens were coated with gold mixture was allowed to stand for 6 min, and then 150 µl and then mounted into SEM FEI (Quanta 200). of 10% AlCl solution was added; after 5  min 0.5 ml of 1  mol/l NaOH solution were added. The final volume of Pathogenicity test the mixture was brought to 2.5 ml with distilled water. Pathogenicity of F. solani was determined on zucchini The absorbance was measured at 510  nm. A calibration plants under greenhouse conditions. To obtain conidial curve of catechin (C) was used to calculate the concen- suspensions, the surface of cultures was scratched with tration of total flavonoids and the results expressed as mg a sterile scalpel and then the plate was washed with CE/ml extract. The total antioxidant capacity (TAC) was sterilized distilled water. The inoculum then filtered by assayed by the popular ferric reducing antioxidant power gauze to eliminate the large fragments of mycelia. The (FRAP) and expressed as µmole Trolox equivalent/g dry fungal spores were counted using a hemocytometer weight. and adjusted to an approximate concentration 1.5 × 10 conidial/ml. Root of zucchini seedlings were dipped in Evaluation of the inhibitory activities of the desert plant conidia suspension while control was carried out by dip- extracts against F. solani ping roots in sterile distilled water. The inoculated plants Inhibition of F. solani radial growth were incubated in greenhouse under 80% relative humid- F. solani mycelial plugs (5  mm), obtained from seven- ity and 30 ± 2  °C. Koch’s postulates were checked under days-old culture, were added at the center of plates controlled conditions (Choi et al. 2015). contain PDA medium (control) or PDA complemented with various amounts of plant extracts. Mycelium radial Collection of plant materials and preparation of extract growth was measured and the inhibitory activity to radial Aerial parts of thirteen desert plants (leaf, petal, pod, growth was calculated relative to the corresponding con- seed and stem) were collected from different explored trol after 14 days. The concentration of extract (mg/ml) sites in Yanbu region deserts, Saudi Arabia, surround- needed to bring about 50% inhibition of fungal linear ing the site; 24°6′ 7.2288′N 38°6′ 37.6524′E. The collected growth. The half maximal inhibitory concentration (IC ) plants were washed with tap water followed by distilled was assessed by linear regression. water and left in shade and air till completely dried. Each plant sample was separately ground into powder In vivo assessment of the antifungal activity of the most for preparation of the extract. Powdered plant samples active extracts were extracted in acetone/methanol (1:1, v/v). After cen- Based on the results of the in  vitro antifungal experi- trifugation at 5000g for 15  min, the supernatant was de- ment, an experiment was conducted to assess the poten- fated with n-hexane and then evaporated to dryness with tiality of the most active extracts in protecting zucchini a rotary evaporator. The obtained pellet was dissolved plants from crown and foot rot disease under greenhouse in methanol and used for the subsequent experiments. conditions. To get seedlings, sterilized seeds were sown Unless stated differently, all chemicals and solvents were on previously sterilized peat for three weeks. Seedlings in analytical grade (Sigma-Aldrich, Milan, Italy). were dipped, by their roots, into the previously prepared conidial suspension 1.5 × 10 conidial/ml of F. solani Assessment of flavonoids, total phenolics and antioxidant before transplanting to pots containing sterilized peat. capacity of the plant extracts Five groups of infected seedlings were prepared, each The crude extract was used for each plant sample to consisted of 50 plants divided into 10 pots (5/pot). Three assess total phenolics by Folin-Ciocalteu method (Kaur days after transplanting the plants in, the first four groups et  al. 2009). Briefly, 1 ml of the diluted plant extract was were treated with extract of Rosmarinus officeniales, Puli- mixed with one ml of 10% Folin-Ciocalteu reagent for caria crispa, Achillea falcata or Haloxylon salicornicum, 3  min. After through, one ml of anhydrous sodium car- while the fifth group was left without treatment with bonate (20%, w/v) was added to the mixture, followed any plant extract. The extracts were applied to the basal by incubation in dark for 30  min at room temperature. stems of the plants at a concentration equal to the I C , The absorbance of the mixture was measured at 650 nm. obtained from the results of the in vitro antifungal assay, The concentration of phenolics was extrapolated from at the rate of 5 ml/plant. To provide a control, other 50 gallic acid (GA) calibration curve and the results were seedlings were dipped into sterilized distilled water. Khalil et al. AMB Express (2023) 13:6 Page 4 of 12 Fig. 1  A Growth of Fusarium solani on potato dextrose agar medium. B Bright field micrograph showing microconidia small, oval shape (arrows); macroconidia large and aseptate, canoe‑shape with 1–3 septae (arrows) C The center and margin color of the reverse petri dish has yellow to tan color. D Bright field micrograph showing terminal and sub ‑terminal chlamidospores. B Bar = 1 mm; D Bar = 2 mm For each plant of the control group, 5ml of solvent was extract and 2% Tween 20 at half-maximal inhibitory con- applied to its basal stem. The pots were kept in the green -centration (IC ) 0.92 mg/ml. Fungal culture was kept at house under 80% relative humidity and 30 ± 2  °C. Four- 25 ± 2  °C under shaking condition (150  rpm) for seven teen days after inoculation, disease symptoms started to days. Same procedure was prepared to use as a control develop on plants. To estimate disease progress, plants with free P. crispa extract. The mycelia were harvested were monitored daily for the presence of the disease from the culture media after incubation period and sep- symptoms. Four weeks after seedling transplantation, arated by centrifugation at 4000×g, 15  min and washed the disease incidence (DI) was calculated as the ratio of twice using 0.1  M phosphate buffer at pH 7.4. For fixa - symptomatic plants relative to the total number of plants tion, the harvested fungal cells were treated by 3% v/v assessed in the experiment. The disease incidence was glutaraldehyde for two hours inside the fume hood. Cells estimated four weeks after seedling transplant. were washed with buffer and then re-fixed with 1% (w/v) osmium tetroxide (osmic acid) at 5  °C for three hours. Transmission electron microscopy (TEM) Consequently, cells were dehydrated gradually in a series According to the results of the in  vitro antifungal assay of ethanol solutions (10%, 20%, 50%, 60%, 70%, 80% and P. crispa was selected for TEM investigation. To evalu- 90%; and 100%). The ultrathin slices (almost 100  nm) ate the antifungal activity of P. crispa extract against F. were obtained using a Reichert-Jung ultramicrotome solani, spore suspension (1 × 10 spores/ml) was mixed with diamond knife. The ultra-sections were put on 200 with 10 ml potato dextrose supplemented with P. crispa mesh copper grids and stained with 4% aqueous uranyl K halil et al. AMB Express (2023) 13:6 Page 5 of 12 Fig. 2 Scanning electron micrographs of F. solani showing A Solitary conidiophore B Chlamidospore C, D Conidiophores with microconidia. A, C, D Bar = 2.5 μm; B Bar = 5 μm Table 1 Total phenolics (mg GAE/ml extract), flavonoids (mg CE/ml extract), total antioxidant capacity (TAC, mM TE/g extract) and antifungal activity (IC , mg/ml) of aqueous methanolic extracts of the different plant species Plant Phenolics Flavonoids TAC IC e* d c f Horwoodia dicksoniae 25.08 ± 0.34 5.14 ± 0.02 0.82 ± .052 1.94 ± 0.05 c c b g Gloiosiphonia capillaris 14.92 ± 0.53 3.60 ± 0.03 0.59 ± 0.03 2.05 ± 0.04 g g d ef Teucrium polium 29.21 ± 0.13 f 11.44 ± 0.1 1.16 ± 0.03 1.75 ± 0.07 a a a ef Agrostis lachnantha 4.96 ± 0.96 1.63 ± 0.01 0.33 ± 0.03 1.89 ± 0.16 h i h d Artemisia sieberia 40.92 ± 2.51 18.19 ± 0.32 2.32 ± 0.13 1.35 ± 0.05 k k i ab Achillea falcata 58.64 ± 0.30 24.70 ± 0.39 2.81 ± 0.015 0.77 ± 0.03 hi i h a Pulicaria crispa 42.31 ± 0.78 17.37 ± 0.22 2.33 ± 0.04 0.67 ± 0.02 f e de de Achillea fragrantissima 28.22 ± 0.13 7.35 ± 0.1 1.29 ± 0.03 1.51 ± 0.06 g f e ef Artemisia Judaica 30.49 ± 0.08 9.74 ± 0.06 1.33 ± 0.03 1.73 ± 0.04 i g i ab Rosmarinus officinalis 44.65 ± 0.7 11.09 ± 0.01 2.80 ± 0.06 0.85 ± 0.02 d b a e Rhazya stricta 18.08 ± 0.04 2.46 ± 0.08 0.38 ± 0.2 1.62 ± 0.03 h h f bc Haloxylon salicornicum 41.22 ± 0.02 14.93 ± 0.12 1.86 ± 0.04 1.08 ± 0.04 j j h d Rhanterium epapposum 47.22 ± 0.13 22.30 ± 0.25 2.40 ± 0.07 1.38 ± 0.03 *Values are mean ± standard error of three independent replicates. Different letters in the same column indicate significant difference (P > 0.05) as analyzed by Duncan test Khalil et al. AMB Express (2023) 13:6 Page 6 of 12 Levels of phenolics, flavonoids and TAC show a great variability among of the tested plant extracts The quantitative evaluation of flavonoids and total poly - phenols exhibited considerable variation in their con- centrations among the aqueous methanolic extracts of various plant species (Table  1). The levels of total poly - phenols ranged from 4.96 to 58.31  mg GAE/ g crude extract and from 1.63 to 24.37  mg CE/g crude extract, respectively. Predictably, the total Phenolic content was higher than that of total flavonoid in all extracts. Achil - lea falcata, member of family Asteraceae, showed the highest levels of total phenolics and flavonoids (Table  1). However, the lowest levels of phenolics (4.96  mg/g) and flavonoids (1.63  mg/g) were detected in A. lachnan - Fig. 3 Hierarchical clustering analysis of total polyphenols, tha that belongs to family Amaranthaceae. TAC of the flavonoids, total antioxidant capacities ( TAC) and antifungal activities tested extracts ranged from 0.33 to 2.8 mM TE/g crude (IC ) of the different plant extracts. The relative values are shown in the heatmap based on the average value (n = 3) for each metabolite. extract. In consistence with the levels of total polyphe- Red and blue colors indicate lower and higher concentrations, nols, TAC of A. falcata extract was 10-fold higher than respectively that of A. lachnantha. These results suggest a clear cor - relation between the amounts of phenolic compounds in the extracts and their TAC. acetate/lead citrate to be ready for a JEOL JEM-1400 The phenolic rich extracts inhibited the hyphal growth transmission electron microscope at an accelerating volt- of the tested F. solani isolate age of 40 to 120 kV. The antimycotic potential of 13 plant extracts were inves - tigated individually against the investigated F. solani Statistical analysis isolate. All the tested extracts showed inhibitory effects Data analyses were performed using Statistical Analy- on the hyphal growth of F. solani (Table  1), but at dis- sis System (SPSS Inc., Chicago, IL, USA). Tukey’s Test parate capabilities. According to the results in Table  1 (p < 0.05) was applied for separations of mean (n = 5). and the hierarchical clustering analysis (HCA, Fig.  3), Cluster analysis was performed by using Pearson dis- the crude extracts could be divided into three groups tance metric of the MultiExperiment Viewer (MeV) 4 with respect to I C values against F. solani. The groups software package (version 4.5, Dana-Farber Cancer Insti- included extracts characterized by: high antifungal activ- tute, Boston, MA, USA). ity (group B; R. officinalis, P. crispa, A. falcata and H. salicornicum); moderate inhibition of F. solani (group Results A; R. epapposum, A. sieberia); low antimycotic poten- Isolation and morphological identification of F. solani tial (group C; H. dicksoniae, G. Capillaris, T. polium, A. In the present study, isolates were obtained from infected lachnantha, A. fragrantissima, A. judaica, R. stricta). zucchini plants, showing colonies that characterized by Indeed, extracts in groups A and B were characterized cream-to-white color with a yellowish center and a cream by the higher phenolic contents, while those in group C or pall yellow reverse on agar (Fig.  1). The Fusarium possessed the lower levels of phenolics. This result is con - septate hyphae produced non-branched conidiophores sistent with the correlation analysis, where positive rela- terminated with two different types of conidia. Depend - tionships were observed between the total phenolics and ing on culture age conidia varied in size. Microconidia flavonoids contents of the extract (0.74 and 0.63, respec - were oval, unicellular, and abundant, while macroconidia tively) and their antifungal activities (Fig.  4). However, were canoe-shaped, multicellular, septate with one to the qualitative variations in the phenolic profiles of the three septa at an average size of 15.7–28.9 × 2.6–3.4  μm. plant extracts and the synergic or antagonistic reactions Chlamydospores were always detected (Figs. 1, 2). The F. cannot be neglected (Nwonuma et al. 2019). For instance, solani were deposited and available in Culture Collection although extracts of P. crispa and A. falcata showed com- Ain-Shams University (Cairo-Egypt), under the numbers parable levels of polyphenols but P. crispa caused higher CCASU-2022-F6. reduction in disease incidence. K halil et al. AMB Express (2023) 13:6 Page 7 of 12 Fig. 4 Correlation between the antifungal activity of the plant extracts and their total phenolic (A) and total flavonoid (B) contents P. crispa, A. falcata, R. officinalis and H. salicornicum extracts P. crispa, A. falcata, R. officinalis and H. salicornicum inhibited the development of crown and foot rot disease against phytopathogens have been rarely investigated, on zucchini plants the application of crude extracts of numerous wild plants Based on the results of the in  vitro antifungal assay, the to reduce the development of plant diseases has been most powerful plant extracts (P. crispa, A. falcata, R. investigated. officinalis and H. salicornicum) were selected to test their influence on the development of Fusarium crown P. crispa extract induced ultrastructure disorders in F. solani and foot rot on zucchini. Interestingly, the four extracts In the present study, transmission electron microscope significantly decreased the disease incidence, where P. (TEM) was employed to understand the ultrastructural crispa was the most effective as it resulted of 34% reduc - mechanisms behind the antifungal capacity of the phe- tion in disease incidence compared to the control (Fig. 5). nolic rich extracts, using P. crispa extract as a repre- Although the in  vivo activities of phenolic extracts of sentative of the most powerful group of extracts (group Khalil et al. AMB Express (2023) 13:6 Page 8 of 12 Fig. 5 Impact of phenolic rich extracts of P. crispa, A. falcata, R. officinalis and H. salicornicum on incidence of Fusarium crown and foot root on zucchini. The different letters above the bars indicate significant differences (P < 0.05) as analyzed by the Tukey’s test B, Fig.  3). The hyphae of F. solani were observed after crops repeatedly leads to high yield worldwide (Savary treatment with the extract of P. crispa in compared to et al. 2019). In fact, rapid and successful treatment of fun- untreated hyphae (control) (Fig.  6A, B). TEM micro- gal infection depends on accurate identification of causa - graphs of hyphal cells of F. solani growing on PDA tive agent as well as selection of the suitable treatment showed a normal, condensed and rigid homogeneous cell strategies. Poletto et  al. (2006) had ability to identify wall with plasma membrane closely appraised against the Fusarium species via morphological characters, follow- cell wall (Fig.  6A). Cytoplasm appeared dense and meta- ing procedure suggested by Ohara and Tsuge (2004). bolically active, determined by the presence of ribosomes There are several factors such as genetic, morphologi - and organelles. Moreover, the large number of organelles cal and environmental conditions have an influence on including mitochondria, vacuoles, nuclei, and endoplas- plant secondary metabolome, including phenolic com- mic reticula found. On the other hand, the observation pounds, at the qualitative and quantitative levels (Cirak of hyphal cells of F. solani amended on medium contain- et al. 2014). Alcoholic extracts of 32 plants displayed high ing the extract of P. crispa showed various degrees of variations in phenolic and flavonoid contents (Wojdyło cell deteriorations (Fig.  6C, D). Obviously, the treatment et  al. 2007). It was reported that there are many varia- disturbed plasmalemma, induced intense of cytoplasmic tions in the levels of the total phenolics and flavonoids vacuolation while organelles became gradually expanded not only among the plant species but also among the dif- to the periphery of the cells (Fig.  6C). Autophagosomes ferent plant organs (Maisuthisakul et al. 2007; Çirak et al. were observed that expanded all over the cells (Fig.  6D). 2011). Eventually, hyphal cells were highly distorted and cru- Plant phenolic compounds possess effective free radical cially damaged where organelles such as mitochondria scavenging activity (Heim et al. 2002; Maisuthisakul et al. and nucleus were no longer discernible. 2007) reported a linear correlation between total phe- The annual increase in the organic food demand due to nolic compounds and TAC. It is worth to mention that consumers awareness of healthy food and environmen- the potentiality of phenolic compounds as a free radical- tal save agriculture drive the researchers to explore the scavenging activity depends on their structure, concen- natural ecosystem searching for an alternative treatment tration and the substitution of hydroxyl groups (Karamac strategy to chemical pesticide or effective agents to act et al. 2005). against phytopathogens. Fungal infection to commercial K halil et al. AMB Express (2023) 13:6 Page 9 of 12 Fig. 6 Transmission electron micrographs of F. solani growing on PDA medium (A, B) or amended with 0.92 mg/ml plant extract (C, D). CW cell wall, V vacuoles, PM plasma membrane, S septum, N nucleus, M mitochondria, C cytoplasm With increasing attention for medicinal and aromatic collected from Al-Qassim region, Saudi Arabia, the plants as an alternative medicine, the bioprospecting for extract of H. salicornicum showed antifungal activities bioactive phytochemicals becomes imperative. In this against Stemphylium botryosum, Alternaria alternata, regard, several reports have pointed to the use of natu- Botrytis cinerea and F. solani (El-Mergawi et  al. 2018). ral products, especially phytochemicals, as antifungal Phenolic rich methanolic leaf extracts of R. officinalis agents (Cowan 1999). The antifungal capacity of phe - collected from Northern Riyadh, Saudi Arabia, showed nolics against plant pathogenic fungi is well recognized significant antimycotic properties against Penicillium (Almuhayawi et al. 2021; Nguyen et al. 2013). In line with ochrochloron, P. funiculosum, Aspergillus niger, A. flavus the present results, previous studies have pointed to the A. ochraceus and Candida albicans (Elansary et al. 2020) . significant role of phenolic compounds and their func - Indeed, the adverse impact of phenolic compounds tional groups in the antifungal activity of the crude plant on fungi depends both on concentration and structure extracts (Wink 2015). The crude extract of P. crispa pre - (Ansari et  al. 2013; Gallucci et  al. 2014). Within this sented strong antifungal activity against Aspergillus niger framework, the quantitative structure-activity relation- and Candida albicans (León et  al. 2014). Although the ship a strong correlation between the antifungal poten- antifungal activity of alcoholic, ethyl acetate and water tial of phenolics and flavonoids and their molecular and extracts of several Achillea sp. has been reported, the structural properties has been revealed (Dambolena et al. antifungal activity of A. falcata phenolic extract is poorly 2012; Gallucci et al. 2014). In terms of their mechanisms investigated (Zengin et  al. 2017). Among 15 wild plants of action, phenolics and flavonoids can exhibit antifungal Khalil et al. AMB Express (2023) 13:6 Page 10 of 12 activity by disrupting cell division, hyphal formation and and Haloxylon salicornicum are phenolic rich plants / or triggering sever oxidative stress leading to cell death that prevent Fusarium crown disease and foot rot (Ansari et al. 2013; Cowan 1999). This may be attribute to caused by F. solani in  vitro and in  vivo. The extracts of the direct impact of active phytochemicals ingredients on these four plants significantly reduced the incidence fungal cells or due to induction of defense mechanisms in of the disease in zucchini. These plant extracts have a the host plant (Al-Wakeel et al. 2013; Ansari et al. 2013). bright future in the field of plant protection to replace For instance, treatment of tomato seed with Origa- traditional synthetic fungicides. num vulgare essential oils reduced the incidence of Fusarium wilt caused by Fusarium oxysporum f.sp. lyco- Abbreviations persici (Gonçalves et  al. 2021). Acetone extracts of Aga- FRAP Ferric reducing antioxidant power panthus caulescens (15  mg/ml), Paenibacillus sp. was MNSA Modified Nash‑Snyder agar PDA Potato dextrose agar reported to significantly inhibit the incidence and sever - SEM Scanning electron microscope ity of black rot, caused by Xanthomonas campestris, on CPD Critical point drying the leaves of Brassica napus (Mandiriza et  al. 2018; Per-GA Gallic acid TCA Total antioxidant capacity saud et  al. 2019) reported that the application of lemon DI Disease incidence grass and thick leaf thyme aqueous extracts have reduced TEM Transmission electron microscope the severity of Sheath blight disease caused by Rhizoc-TAC Total antioxidant capacity HCA Hierarchical clustering analysis tonia solani in rice. Phenolic extracts of Orobanche cre- nata and Sanguisorba minor inhibited the rot of sweet Acknowledgements cherry fruits (Gatto et al. 2016). Xerophyte plants showed Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank abroad range of polyphenols including phenolic acids, (EKB). flavonoids, lignans and coumarins that have an antifungal activity against most of soilborne pathogens and success- Author contributions The manuscript conceptualization, MSMM and AMS; methodology, AMAK, fully applied as fungicides. AMS, MSMM and SMSA; data analysis, AMAK and AMS writing the original draft The effects of crude medicinal plant extracts on fun - preparation, AMS and AMAK; The manuscript review and editing, AMAK, AMS, gal development, including conidial germination were MSMM and SMSA; All authors have read and agreed to the published version of the manuscript. investigated (Khalil and Hashem 2018; Niño et  al. 2012; Ren et al. 2012). Moreover, phenolic compounds includ- Funding ing cinnamic, gallic, vanillic, salicylic and ferulic acids are Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank reported to induce collapse and shrinkage of the bacte- (EKB). This research received no external funding. rial and fungal cells (Akter et al. 2019; Nguyen et al. 2013; Abdelmohsen et  al. 2020). Supporting this hypothesis, Availability of data and materials Not applicable. when some plant extracts applied on Fusarium species hyphal cells showed similar signs (Benhamou and Thé - Declarations riault 1992). Moreover, some flavonoids such as querce - tin and luteolin were induced some morphological and Ethics approval and consent to participate ultrastructural damages of the tested fungal species (Bái- Not applicable. dez et al. 2006). In line with this concept, the presence of Consent for publication high content of phenolics and flavonoids in plant extracts All authors agree to submit this manuscript for publication. enable them to disturb the cell permeability, by their lipo- Competing interests philic effects, and uncouple the oxidative phosphoryla - Authors have no competing interests to declare. tion due to their hydroxyl groups (Dambolena et al. 2012; Gallucci et al. 2014). It is known that Macroautophagy or Received: 10 September 2022 Accepted: 9 January 2023 type II programmed cell death of fungal cells is activated by constituents of growth medium and associated with cellular stress (Hashem et  al. 2020; Khalil et  al. 2019). 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Plants from a semi-arid environment as a source of phytochemicals against Fusarium crown and foot rot in zucchini

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Abstract

Fusarium crown and foot rot, caused by F. solani f. sp. cucurbitae, are major fungal diseases affecting zucchini and other cucurbits. Despite the efficacy of synthetic fungicides, their health and environmental hazards have highlighted the urgent need for safer alternatives, such as phytochemical‑based biocides. Owing to the upregulation of the plant sec‑ ondary metabolism under stressful conditions, bioprospecting in harsh environments could reveal ore plants for bio‑ active metabolites. In this study, thirteen wild plants were collected from their natural habitat in a semiarid environ‑ ment (Yanbu, Saudi Arabia) and extracted to obtain phenolics rich extracts. Total polyphenols, flavonoids, antioxidant capacities and the antifungal activities of the extracts against a pathogenic isolate of F. solani were assessed. Fusarium solani was isolated from infected zucchini and characterized by scanning electron microscopy. Hierarchical clustering analysis of the phytochemical screening and in vitro bioactivity revealed that Rosmarinus officinalis, Pulicaria crispa, Achillea falcata and Haloxylon salicornicum were the richest in polyphenols and the most powerful against F. solani. Further, the extracts of these four plants significantly decreased the disease incidence in zucchini, where P. crispa was the premier. Interestingly, results of transmission electron microscopy revealed that extract of P. crispa, as a representa‑ tive of the powerful group, induced ultrastructural disorders in fungal cells. Therefore, this study suggests the use of R. officinalis, P. crispa, A. falcata and H. salicornicum grown in semi‑arid environments as ore plants to develop phyto ‑ chemical‑based biocides against Fusarium crown and foot rot. Keywords Phytochemicals, Polyphenols, Biocides, Fusarium solani, Zucchini, Ultrastructure a broad range of host plants (Barreto et  al. 2003), and Introduction attacks the postharvest crops (Akrami et  al. 2012). The Fusarium species are the most common soil-born fungi; diseases caused by F. solani are characterized by many despite being saprophytes, they are well-known as plant symptoms such as root and stem rotting, wilting, leaf yel- pathogens (Coleman 2016). Fusarium solani is a cosmo- lowing, and sudden death (Nemec et al. 1976). Although politan aggressive plant pathogenic fungus that invades the persistence of F. solani in the agricultural soil after disease occurrence is poorly investigated, some formae speciales, such as F. solani f. sp. cucurbitae and fragariae, Ahmed M. A. Khalil and Ahmed M. Saleh have contributed equally to this manuscript and share first authorship have been reported to form chlamydospores that confer survival of phytopathogenic soil borne population after *Correspondence: Mahmoud S. M. Mohamed tillage (Henry et al. 2019). msaleh@sci.cu.edu.eg Many researchers demonstrated that F. solani f. Botany and Microbiology Department, Faculty of Science, Al‑Azhar sp. cucurbitae has host specificity for Cucurbitaceae University, Cairo, Egypt Department of Botany and Microbiology, Faculty of Science, Cairo (Boughalleb et  al. 2007; Paternotte 1987). This phy - University, Giza 12613, Egypt topathogen has been recorded causing disease in field © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Khalil et al. AMB Express (2023) 13:6 Page 2 of 12 production in pumpkin, muskmelon, cucumber, water- compounds have been suggested to attend as valuable melon, and rootstock hybrids in addition to be the causal substitutes to the chemical control of plant pathogens in agent of crown rot for several cucurbits including Zuc- agricultural soils (Langcake 1981). chini squash (Pérez-Hernández et  al. 2017). Plant resist- From a physiological point of view, the production of ance to this pathogen stills limited, while the most biologically active phytochemicals has been reported to common control procedures for the disease caused by be upregulated under adverse environmental conditions F. solani f. sp. cucurbitae are the rotation with non-host (Oh et al. 2009). In this context, due to their severe habi- crops, preventive and curative fungicide application. tat characterized by little water and high temperature, From the human health point of view, application of fun- xerophytic plants have been reported to hyperaccumu- gicides is undesirable as low concentrations of synthetic late phenolic compounds, rendering them a promising pesticides in agricultural products might cause adverse source for antifungal phytochemicals (Fahn and Cutler health effects (Akoto et al. 2013). Further, the accumula - 1992; Yehia et  al. 2020). For instance, Horwoodia dick- tion of pesticides in the agriculture soil may lead to irre- soniae extracts revealed growth inhibitory effect against versible environmental damages such as (i) water-table Aspergillus fumigatus, Streptococcus pneumoniae and and water body contamination, (ii) imbalance in biologi- Escherichia coli (Abdelwahab et  al. 2016). Moreover, the cal diversity, (iii) reduce efficiency of microorganisms antifungal activity of Rhazya stricta extract was per- to degrade pesticides and (iv) develop resistant strains formed against different fungal species including Tricho - (Boughalleb et al. 2007; Abdelkader et al. 2022). phyton longifusis, Aspergillus flavus, Candida albicans Despite the worthwhile results of synthetic fungicides and F. solani. It showed a good inhibitory effect with against F. solani, there is urgent need for developing reducing the hyphal growth of all investigated fungal spe- more safe controlling methods, both for human health cies (Khan and Khan 2007). and environment (Chandel and Deepika 2010; Mostafa Therefore, this study aims to bioprospect in a semi- et  al. 2009). In this regard, the application of plant bio- arid environment (at Yanbu desert, Saudi Arabia) for active molecules and phytochemicals could provide an xerophytes rich in antifungal phenolics, with respect to effectual alternative for synthetic fungicides (Cowan F. solani. Thirteen plants were collected and extracted to 1999; Al Kashgry et  al. 2020). Among these, phenolic obtain phenolics rich extracts. compounds, the most diverse group of plant secondary metabolites including simple phenols, phenolic acids, Materials and methods complex tannins, flavonols and dihydrochalcones, are Isolation and identification of Fusarium solani proved as defensive agents against plant pathogens. Their Fusarium solani was isolated in the laboratory from main advantage is to be environmentally safe as they are infected basal stems of zucchini plants (Cucurbita pepo) easily biodegraded (Steinkellner and Mammerler 2007). displaying external signs of zucchini wilt and crown rot. Several phenolics are reported to accumulate in plant tis- Fusarium solani isolation was achieved by cutting the sues constitutively (i.e. phytoanticipins) and also hyper- infected basal stem into small pieces ranging from 2 to accumulated in response to pathogens challenge (i.e. 3  mm. The pieces were surface disinfected with sodium phytoalexins), playing direct and indirect role in disease hypochlorite (10%) solution for 2 min, washed with ster- resistance (Akhtar and Malik 2000; Lattanzio et al. 2006). ile distilled water several times. Pieces were transferred Generally, antifungal phenolics could act directly by dis- aseptically onto Fusarium selective medium called modi- rupting the fungal morphology, physiology and ultra- fied Nash-Snyder agar (MNSA) (1  g/l KH PO , 0.5  g/l 2 4 structure (Báidez et  al. 2006; Cowan 1999; Khalil et  al. MgSO -7H O, 15  g/l peptone, 20  g/ Agar, 1  g/l penta- 4 2 2020; Nguyen et al. 2013) or indirectly by stimulating the chloronitobenzen, 0.3  g/l streptomycin sulfate, 0.12  g/l plant defense system (Al-Wakeel et al. 2013). The delete - neomycin sulfate) (Nash and Snyder 1962) and incu- rious action of phenolics against fungi could be ascribed bated at 26 ± 2  °C for 5 days. The fungal hyphae were to the presence of acidic hydroxyl group attached to aro- transferred to petri dishes containing potato dextrose matic ring that have lipophilic properties allowing phe- agar medium (PDA). Morphological characteristics were nolic compounds to penetrate the plasma membrane identified both on Czapek-Dox agar (30  g/l sucrose, making ionic homeostatic disturbance (Dambolena et al. 3 g/l NaNO , 0.5  g/l KCl, 100  mg/l F eSO -7H O, 0.5  g/l 3 4 2 2012; Gallucci et al. 2014). Further, these hydroxyl groups MgSO -7H O, 1  g/l K HPO ) and potato dextrose agar 4 2 2 4 could inhibit coupling between the electron transport (PDA) medium as well as microscopic examination and phosphorylation reactions (Parvez et  al. 2004). (Booth 1977; Ellis 1976; Raper and Fennell 1965; Raper Moreover, phenolics have been reported to interfere with and Thom 1949; Rifai 1969) To confirm morphologi - the key enzymes that regulate fungal growth and devel- cal identification, Scanning electron microscopy (SEM) opment (Schwalb and Feucht 1999). Therefore, phenolic was carried out. It was accomplished to approve the K halil et al. AMB Express (2023) 13:6 Page 3 of 12 morphological characteristics of fungal species. A small expressed as mg GAE/ml extract. Total flavonoids con - sample of fungal colony was immersed in glutaralde- tents were assayed by the AlCl colorimetric method hyde (2.5%) for 15 min. The samples were dehydrated by (Sakanaka et  al. 2005). Briefly, 50  µl of each crude plant ethanol-acetone gradient. To avoid collapse in SEM the extract was mixed up to 1.45 ml of distilled water, fol- critical point drying (CPD) was used. Using an Emitech lowed by addition of 75  µl of 5% NaNO solution. The K550X coating unit, the specimens were coated with gold mixture was allowed to stand for 6 min, and then 150 µl and then mounted into SEM FEI (Quanta 200). of 10% AlCl solution was added; after 5  min 0.5 ml of 1  mol/l NaOH solution were added. The final volume of Pathogenicity test the mixture was brought to 2.5 ml with distilled water. Pathogenicity of F. solani was determined on zucchini The absorbance was measured at 510  nm. A calibration plants under greenhouse conditions. To obtain conidial curve of catechin (C) was used to calculate the concen- suspensions, the surface of cultures was scratched with tration of total flavonoids and the results expressed as mg a sterile scalpel and then the plate was washed with CE/ml extract. The total antioxidant capacity (TAC) was sterilized distilled water. The inoculum then filtered by assayed by the popular ferric reducing antioxidant power gauze to eliminate the large fragments of mycelia. The (FRAP) and expressed as µmole Trolox equivalent/g dry fungal spores were counted using a hemocytometer weight. and adjusted to an approximate concentration 1.5 × 10 conidial/ml. Root of zucchini seedlings were dipped in Evaluation of the inhibitory activities of the desert plant conidia suspension while control was carried out by dip- extracts against F. solani ping roots in sterile distilled water. The inoculated plants Inhibition of F. solani radial growth were incubated in greenhouse under 80% relative humid- F. solani mycelial plugs (5  mm), obtained from seven- ity and 30 ± 2  °C. Koch’s postulates were checked under days-old culture, were added at the center of plates controlled conditions (Choi et al. 2015). contain PDA medium (control) or PDA complemented with various amounts of plant extracts. Mycelium radial Collection of plant materials and preparation of extract growth was measured and the inhibitory activity to radial Aerial parts of thirteen desert plants (leaf, petal, pod, growth was calculated relative to the corresponding con- seed and stem) were collected from different explored trol after 14 days. The concentration of extract (mg/ml) sites in Yanbu region deserts, Saudi Arabia, surround- needed to bring about 50% inhibition of fungal linear ing the site; 24°6′ 7.2288′N 38°6′ 37.6524′E. The collected growth. The half maximal inhibitory concentration (IC ) plants were washed with tap water followed by distilled was assessed by linear regression. water and left in shade and air till completely dried. Each plant sample was separately ground into powder In vivo assessment of the antifungal activity of the most for preparation of the extract. Powdered plant samples active extracts were extracted in acetone/methanol (1:1, v/v). After cen- Based on the results of the in  vitro antifungal experi- trifugation at 5000g for 15  min, the supernatant was de- ment, an experiment was conducted to assess the poten- fated with n-hexane and then evaporated to dryness with tiality of the most active extracts in protecting zucchini a rotary evaporator. The obtained pellet was dissolved plants from crown and foot rot disease under greenhouse in methanol and used for the subsequent experiments. conditions. To get seedlings, sterilized seeds were sown Unless stated differently, all chemicals and solvents were on previously sterilized peat for three weeks. Seedlings in analytical grade (Sigma-Aldrich, Milan, Italy). were dipped, by their roots, into the previously prepared conidial suspension 1.5 × 10 conidial/ml of F. solani Assessment of flavonoids, total phenolics and antioxidant before transplanting to pots containing sterilized peat. capacity of the plant extracts Five groups of infected seedlings were prepared, each The crude extract was used for each plant sample to consisted of 50 plants divided into 10 pots (5/pot). Three assess total phenolics by Folin-Ciocalteu method (Kaur days after transplanting the plants in, the first four groups et  al. 2009). Briefly, 1 ml of the diluted plant extract was were treated with extract of Rosmarinus officeniales, Puli- mixed with one ml of 10% Folin-Ciocalteu reagent for caria crispa, Achillea falcata or Haloxylon salicornicum, 3  min. After through, one ml of anhydrous sodium car- while the fifth group was left without treatment with bonate (20%, w/v) was added to the mixture, followed any plant extract. The extracts were applied to the basal by incubation in dark for 30  min at room temperature. stems of the plants at a concentration equal to the I C , The absorbance of the mixture was measured at 650 nm. obtained from the results of the in vitro antifungal assay, The concentration of phenolics was extrapolated from at the rate of 5 ml/plant. To provide a control, other 50 gallic acid (GA) calibration curve and the results were seedlings were dipped into sterilized distilled water. Khalil et al. AMB Express (2023) 13:6 Page 4 of 12 Fig. 1  A Growth of Fusarium solani on potato dextrose agar medium. B Bright field micrograph showing microconidia small, oval shape (arrows); macroconidia large and aseptate, canoe‑shape with 1–3 septae (arrows) C The center and margin color of the reverse petri dish has yellow to tan color. D Bright field micrograph showing terminal and sub ‑terminal chlamidospores. B Bar = 1 mm; D Bar = 2 mm For each plant of the control group, 5ml of solvent was extract and 2% Tween 20 at half-maximal inhibitory con- applied to its basal stem. The pots were kept in the green -centration (IC ) 0.92 mg/ml. Fungal culture was kept at house under 80% relative humidity and 30 ± 2  °C. Four- 25 ± 2  °C under shaking condition (150  rpm) for seven teen days after inoculation, disease symptoms started to days. Same procedure was prepared to use as a control develop on plants. To estimate disease progress, plants with free P. crispa extract. The mycelia were harvested were monitored daily for the presence of the disease from the culture media after incubation period and sep- symptoms. Four weeks after seedling transplantation, arated by centrifugation at 4000×g, 15  min and washed the disease incidence (DI) was calculated as the ratio of twice using 0.1  M phosphate buffer at pH 7.4. For fixa - symptomatic plants relative to the total number of plants tion, the harvested fungal cells were treated by 3% v/v assessed in the experiment. The disease incidence was glutaraldehyde for two hours inside the fume hood. Cells estimated four weeks after seedling transplant. were washed with buffer and then re-fixed with 1% (w/v) osmium tetroxide (osmic acid) at 5  °C for three hours. Transmission electron microscopy (TEM) Consequently, cells were dehydrated gradually in a series According to the results of the in  vitro antifungal assay of ethanol solutions (10%, 20%, 50%, 60%, 70%, 80% and P. crispa was selected for TEM investigation. To evalu- 90%; and 100%). The ultrathin slices (almost 100  nm) ate the antifungal activity of P. crispa extract against F. were obtained using a Reichert-Jung ultramicrotome solani, spore suspension (1 × 10 spores/ml) was mixed with diamond knife. The ultra-sections were put on 200 with 10 ml potato dextrose supplemented with P. crispa mesh copper grids and stained with 4% aqueous uranyl K halil et al. AMB Express (2023) 13:6 Page 5 of 12 Fig. 2 Scanning electron micrographs of F. solani showing A Solitary conidiophore B Chlamidospore C, D Conidiophores with microconidia. A, C, D Bar = 2.5 μm; B Bar = 5 μm Table 1 Total phenolics (mg GAE/ml extract), flavonoids (mg CE/ml extract), total antioxidant capacity (TAC, mM TE/g extract) and antifungal activity (IC , mg/ml) of aqueous methanolic extracts of the different plant species Plant Phenolics Flavonoids TAC IC e* d c f Horwoodia dicksoniae 25.08 ± 0.34 5.14 ± 0.02 0.82 ± .052 1.94 ± 0.05 c c b g Gloiosiphonia capillaris 14.92 ± 0.53 3.60 ± 0.03 0.59 ± 0.03 2.05 ± 0.04 g g d ef Teucrium polium 29.21 ± 0.13 f 11.44 ± 0.1 1.16 ± 0.03 1.75 ± 0.07 a a a ef Agrostis lachnantha 4.96 ± 0.96 1.63 ± 0.01 0.33 ± 0.03 1.89 ± 0.16 h i h d Artemisia sieberia 40.92 ± 2.51 18.19 ± 0.32 2.32 ± 0.13 1.35 ± 0.05 k k i ab Achillea falcata 58.64 ± 0.30 24.70 ± 0.39 2.81 ± 0.015 0.77 ± 0.03 hi i h a Pulicaria crispa 42.31 ± 0.78 17.37 ± 0.22 2.33 ± 0.04 0.67 ± 0.02 f e de de Achillea fragrantissima 28.22 ± 0.13 7.35 ± 0.1 1.29 ± 0.03 1.51 ± 0.06 g f e ef Artemisia Judaica 30.49 ± 0.08 9.74 ± 0.06 1.33 ± 0.03 1.73 ± 0.04 i g i ab Rosmarinus officinalis 44.65 ± 0.7 11.09 ± 0.01 2.80 ± 0.06 0.85 ± 0.02 d b a e Rhazya stricta 18.08 ± 0.04 2.46 ± 0.08 0.38 ± 0.2 1.62 ± 0.03 h h f bc Haloxylon salicornicum 41.22 ± 0.02 14.93 ± 0.12 1.86 ± 0.04 1.08 ± 0.04 j j h d Rhanterium epapposum 47.22 ± 0.13 22.30 ± 0.25 2.40 ± 0.07 1.38 ± 0.03 *Values are mean ± standard error of three independent replicates. Different letters in the same column indicate significant difference (P > 0.05) as analyzed by Duncan test Khalil et al. AMB Express (2023) 13:6 Page 6 of 12 Levels of phenolics, flavonoids and TAC show a great variability among of the tested plant extracts The quantitative evaluation of flavonoids and total poly - phenols exhibited considerable variation in their con- centrations among the aqueous methanolic extracts of various plant species (Table  1). The levels of total poly - phenols ranged from 4.96 to 58.31  mg GAE/ g crude extract and from 1.63 to 24.37  mg CE/g crude extract, respectively. Predictably, the total Phenolic content was higher than that of total flavonoid in all extracts. Achil - lea falcata, member of family Asteraceae, showed the highest levels of total phenolics and flavonoids (Table  1). However, the lowest levels of phenolics (4.96  mg/g) and flavonoids (1.63  mg/g) were detected in A. lachnan - Fig. 3 Hierarchical clustering analysis of total polyphenols, tha that belongs to family Amaranthaceae. TAC of the flavonoids, total antioxidant capacities ( TAC) and antifungal activities tested extracts ranged from 0.33 to 2.8 mM TE/g crude (IC ) of the different plant extracts. The relative values are shown in the heatmap based on the average value (n = 3) for each metabolite. extract. In consistence with the levels of total polyphe- Red and blue colors indicate lower and higher concentrations, nols, TAC of A. falcata extract was 10-fold higher than respectively that of A. lachnantha. These results suggest a clear cor - relation between the amounts of phenolic compounds in the extracts and their TAC. acetate/lead citrate to be ready for a JEOL JEM-1400 The phenolic rich extracts inhibited the hyphal growth transmission electron microscope at an accelerating volt- of the tested F. solani isolate age of 40 to 120 kV. The antimycotic potential of 13 plant extracts were inves - tigated individually against the investigated F. solani Statistical analysis isolate. All the tested extracts showed inhibitory effects Data analyses were performed using Statistical Analy- on the hyphal growth of F. solani (Table  1), but at dis- sis System (SPSS Inc., Chicago, IL, USA). Tukey’s Test parate capabilities. According to the results in Table  1 (p < 0.05) was applied for separations of mean (n = 5). and the hierarchical clustering analysis (HCA, Fig.  3), Cluster analysis was performed by using Pearson dis- the crude extracts could be divided into three groups tance metric of the MultiExperiment Viewer (MeV) 4 with respect to I C values against F. solani. The groups software package (version 4.5, Dana-Farber Cancer Insti- included extracts characterized by: high antifungal activ- tute, Boston, MA, USA). ity (group B; R. officinalis, P. crispa, A. falcata and H. salicornicum); moderate inhibition of F. solani (group Results A; R. epapposum, A. sieberia); low antimycotic poten- Isolation and morphological identification of F. solani tial (group C; H. dicksoniae, G. Capillaris, T. polium, A. In the present study, isolates were obtained from infected lachnantha, A. fragrantissima, A. judaica, R. stricta). zucchini plants, showing colonies that characterized by Indeed, extracts in groups A and B were characterized cream-to-white color with a yellowish center and a cream by the higher phenolic contents, while those in group C or pall yellow reverse on agar (Fig.  1). The Fusarium possessed the lower levels of phenolics. This result is con - septate hyphae produced non-branched conidiophores sistent with the correlation analysis, where positive rela- terminated with two different types of conidia. Depend - tionships were observed between the total phenolics and ing on culture age conidia varied in size. Microconidia flavonoids contents of the extract (0.74 and 0.63, respec - were oval, unicellular, and abundant, while macroconidia tively) and their antifungal activities (Fig.  4). However, were canoe-shaped, multicellular, septate with one to the qualitative variations in the phenolic profiles of the three septa at an average size of 15.7–28.9 × 2.6–3.4  μm. plant extracts and the synergic or antagonistic reactions Chlamydospores were always detected (Figs. 1, 2). The F. cannot be neglected (Nwonuma et al. 2019). For instance, solani were deposited and available in Culture Collection although extracts of P. crispa and A. falcata showed com- Ain-Shams University (Cairo-Egypt), under the numbers parable levels of polyphenols but P. crispa caused higher CCASU-2022-F6. reduction in disease incidence. K halil et al. AMB Express (2023) 13:6 Page 7 of 12 Fig. 4 Correlation between the antifungal activity of the plant extracts and their total phenolic (A) and total flavonoid (B) contents P. crispa, A. falcata, R. officinalis and H. salicornicum extracts P. crispa, A. falcata, R. officinalis and H. salicornicum inhibited the development of crown and foot rot disease against phytopathogens have been rarely investigated, on zucchini plants the application of crude extracts of numerous wild plants Based on the results of the in  vitro antifungal assay, the to reduce the development of plant diseases has been most powerful plant extracts (P. crispa, A. falcata, R. investigated. officinalis and H. salicornicum) were selected to test their influence on the development of Fusarium crown P. crispa extract induced ultrastructure disorders in F. solani and foot rot on zucchini. Interestingly, the four extracts In the present study, transmission electron microscope significantly decreased the disease incidence, where P. (TEM) was employed to understand the ultrastructural crispa was the most effective as it resulted of 34% reduc - mechanisms behind the antifungal capacity of the phe- tion in disease incidence compared to the control (Fig. 5). nolic rich extracts, using P. crispa extract as a repre- Although the in  vivo activities of phenolic extracts of sentative of the most powerful group of extracts (group Khalil et al. AMB Express (2023) 13:6 Page 8 of 12 Fig. 5 Impact of phenolic rich extracts of P. crispa, A. falcata, R. officinalis and H. salicornicum on incidence of Fusarium crown and foot root on zucchini. The different letters above the bars indicate significant differences (P < 0.05) as analyzed by the Tukey’s test B, Fig.  3). The hyphae of F. solani were observed after crops repeatedly leads to high yield worldwide (Savary treatment with the extract of P. crispa in compared to et al. 2019). In fact, rapid and successful treatment of fun- untreated hyphae (control) (Fig.  6A, B). TEM micro- gal infection depends on accurate identification of causa - graphs of hyphal cells of F. solani growing on PDA tive agent as well as selection of the suitable treatment showed a normal, condensed and rigid homogeneous cell strategies. Poletto et  al. (2006) had ability to identify wall with plasma membrane closely appraised against the Fusarium species via morphological characters, follow- cell wall (Fig.  6A). Cytoplasm appeared dense and meta- ing procedure suggested by Ohara and Tsuge (2004). bolically active, determined by the presence of ribosomes There are several factors such as genetic, morphologi - and organelles. Moreover, the large number of organelles cal and environmental conditions have an influence on including mitochondria, vacuoles, nuclei, and endoplas- plant secondary metabolome, including phenolic com- mic reticula found. On the other hand, the observation pounds, at the qualitative and quantitative levels (Cirak of hyphal cells of F. solani amended on medium contain- et al. 2014). Alcoholic extracts of 32 plants displayed high ing the extract of P. crispa showed various degrees of variations in phenolic and flavonoid contents (Wojdyło cell deteriorations (Fig.  6C, D). Obviously, the treatment et  al. 2007). It was reported that there are many varia- disturbed plasmalemma, induced intense of cytoplasmic tions in the levels of the total phenolics and flavonoids vacuolation while organelles became gradually expanded not only among the plant species but also among the dif- to the periphery of the cells (Fig.  6C). Autophagosomes ferent plant organs (Maisuthisakul et al. 2007; Çirak et al. were observed that expanded all over the cells (Fig.  6D). 2011). Eventually, hyphal cells were highly distorted and cru- Plant phenolic compounds possess effective free radical cially damaged where organelles such as mitochondria scavenging activity (Heim et al. 2002; Maisuthisakul et al. and nucleus were no longer discernible. 2007) reported a linear correlation between total phe- The annual increase in the organic food demand due to nolic compounds and TAC. It is worth to mention that consumers awareness of healthy food and environmen- the potentiality of phenolic compounds as a free radical- tal save agriculture drive the researchers to explore the scavenging activity depends on their structure, concen- natural ecosystem searching for an alternative treatment tration and the substitution of hydroxyl groups (Karamac strategy to chemical pesticide or effective agents to act et al. 2005). against phytopathogens. Fungal infection to commercial K halil et al. AMB Express (2023) 13:6 Page 9 of 12 Fig. 6 Transmission electron micrographs of F. solani growing on PDA medium (A, B) or amended with 0.92 mg/ml plant extract (C, D). CW cell wall, V vacuoles, PM plasma membrane, S septum, N nucleus, M mitochondria, C cytoplasm With increasing attention for medicinal and aromatic collected from Al-Qassim region, Saudi Arabia, the plants as an alternative medicine, the bioprospecting for extract of H. salicornicum showed antifungal activities bioactive phytochemicals becomes imperative. In this against Stemphylium botryosum, Alternaria alternata, regard, several reports have pointed to the use of natu- Botrytis cinerea and F. solani (El-Mergawi et  al. 2018). ral products, especially phytochemicals, as antifungal Phenolic rich methanolic leaf extracts of R. officinalis agents (Cowan 1999). The antifungal capacity of phe - collected from Northern Riyadh, Saudi Arabia, showed nolics against plant pathogenic fungi is well recognized significant antimycotic properties against Penicillium (Almuhayawi et al. 2021; Nguyen et al. 2013). In line with ochrochloron, P. funiculosum, Aspergillus niger, A. flavus the present results, previous studies have pointed to the A. ochraceus and Candida albicans (Elansary et al. 2020) . significant role of phenolic compounds and their func - Indeed, the adverse impact of phenolic compounds tional groups in the antifungal activity of the crude plant on fungi depends both on concentration and structure extracts (Wink 2015). The crude extract of P. crispa pre - (Ansari et  al. 2013; Gallucci et  al. 2014). Within this sented strong antifungal activity against Aspergillus niger framework, the quantitative structure-activity relation- and Candida albicans (León et  al. 2014). Although the ship a strong correlation between the antifungal poten- antifungal activity of alcoholic, ethyl acetate and water tial of phenolics and flavonoids and their molecular and extracts of several Achillea sp. has been reported, the structural properties has been revealed (Dambolena et al. antifungal activity of A. falcata phenolic extract is poorly 2012; Gallucci et al. 2014). In terms of their mechanisms investigated (Zengin et  al. 2017). Among 15 wild plants of action, phenolics and flavonoids can exhibit antifungal Khalil et al. AMB Express (2023) 13:6 Page 10 of 12 activity by disrupting cell division, hyphal formation and and Haloxylon salicornicum are phenolic rich plants / or triggering sever oxidative stress leading to cell death that prevent Fusarium crown disease and foot rot (Ansari et al. 2013; Cowan 1999). This may be attribute to caused by F. solani in  vitro and in  vivo. The extracts of the direct impact of active phytochemicals ingredients on these four plants significantly reduced the incidence fungal cells or due to induction of defense mechanisms in of the disease in zucchini. These plant extracts have a the host plant (Al-Wakeel et al. 2013; Ansari et al. 2013). bright future in the field of plant protection to replace For instance, treatment of tomato seed with Origa- traditional synthetic fungicides. num vulgare essential oils reduced the incidence of Fusarium wilt caused by Fusarium oxysporum f.sp. lyco- Abbreviations persici (Gonçalves et  al. 2021). Acetone extracts of Aga- FRAP Ferric reducing antioxidant power panthus caulescens (15  mg/ml), Paenibacillus sp. was MNSA Modified Nash‑Snyder agar PDA Potato dextrose agar reported to significantly inhibit the incidence and sever - SEM Scanning electron microscope ity of black rot, caused by Xanthomonas campestris, on CPD Critical point drying the leaves of Brassica napus (Mandiriza et  al. 2018; Per-GA Gallic acid TCA Total antioxidant capacity saud et  al. 2019) reported that the application of lemon DI Disease incidence grass and thick leaf thyme aqueous extracts have reduced TEM Transmission electron microscope the severity of Sheath blight disease caused by Rhizoc-TAC Total antioxidant capacity HCA Hierarchical clustering analysis tonia solani in rice. Phenolic extracts of Orobanche cre- nata and Sanguisorba minor inhibited the rot of sweet Acknowledgements cherry fruits (Gatto et al. 2016). Xerophyte plants showed Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank abroad range of polyphenols including phenolic acids, (EKB). flavonoids, lignans and coumarins that have an antifungal activity against most of soilborne pathogens and success- Author contributions The manuscript conceptualization, MSMM and AMS; methodology, AMAK, fully applied as fungicides. AMS, MSMM and SMSA; data analysis, AMAK and AMS writing the original draft The effects of crude medicinal plant extracts on fun - preparation, AMS and AMAK; The manuscript review and editing, AMAK, AMS, gal development, including conidial germination were MSMM and SMSA; All authors have read and agreed to the published version of the manuscript. investigated (Khalil and Hashem 2018; Niño et  al. 2012; Ren et al. 2012). Moreover, phenolic compounds includ- Funding ing cinnamic, gallic, vanillic, salicylic and ferulic acids are Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank reported to induce collapse and shrinkage of the bacte- (EKB). This research received no external funding. rial and fungal cells (Akter et al. 2019; Nguyen et al. 2013; Abdelmohsen et  al. 2020). Supporting this hypothesis, Availability of data and materials Not applicable. when some plant extracts applied on Fusarium species hyphal cells showed similar signs (Benhamou and Thé - Declarations riault 1992). Moreover, some flavonoids such as querce - tin and luteolin were induced some morphological and Ethics approval and consent to participate ultrastructural damages of the tested fungal species (Bái- Not applicable. dez et al. 2006). In line with this concept, the presence of Consent for publication high content of phenolics and flavonoids in plant extracts All authors agree to submit this manuscript for publication. enable them to disturb the cell permeability, by their lipo- Competing interests philic effects, and uncouple the oxidative phosphoryla - Authors have no competing interests to declare. tion due to their hydroxyl groups (Dambolena et al. 2012; Gallucci et al. 2014). It is known that Macroautophagy or Received: 10 September 2022 Accepted: 9 January 2023 type II programmed cell death of fungal cells is activated by constituents of growth medium and associated with cellular stress (Hashem et  al. 2020; Khalil et  al. 2019). 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Journal

AMB Express – Springer Journals

Published: Jan 17, 2023

Keywords: Phytochemicals; Polyphenols; Biocides; Fusarium solani; Zucchini; Ultrastructure

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