Antimicrobial Activity of Pinus Wallachiana Against Fusarium Oxysporum f. sp. Cubense and Analysis of its Fractions by HPLC


 Fusarium wilt has ruined banana production and poses a major threat to its industry because of highly virulent Fusarium oxysporum f. sp. cubense (Foc) race 4. The present study focused on the efficacy of Pinus wallachiana and its organic fractions against Foc in in vitro and greenhouse experiments. The presence of polyphenols in the fractions was also investigated using High Performance Liquid Chromatography (HPLC). The in vitro tests carried out for the leaf extract of P. wallachiana showed its inhibitory effect on the mycelial growth and based on this evidence, further characterization of fractions were done. Complete mycelial inhibition and the highest zone of inhibition against Foc was observed for the n-butanol fraction in vitro, while the n-hexane and dichloromethane fractions showed lower disease severity index (DSI) in greenhouse experiments. The fractions were further analysed by HPLC using nine polyphenolic standards, namely quercitin, myrecitin, kaempferol, rutin, gallic acid, trans-ferulic acid, coumeric acid, epicatechin and catechin. The highest content of polyphenols, based on standards used, was quantified in the n-butanol fraction followed by the ethyl acetate fraction of the leaf extract. This is the first report of antimicrobial activity of Pinus wallachiana against Foc to the best of our knowledge.


Introduction
Banana (Musa spp.) is tremendously important for millions of cultivators and corporate growers, both for export and subsistence. The yield of commercial bananas across the world is staggeringly affected by Fusarium wilt (Panama disease) of bananas. It is a soil-borne disease whose causative agent is a hyphomycete i.e. Fusarium oxysporum f. sp. cubense [1][2][3] . Obliteration of Gros Michel by Foc race 1 led to its substitution with resistant Cavendish cultivars that are now susceptible to Foc race 4 speci cally Foc TR4 which gained an emplacement from South East Asia to Africa and recently entrenched in Latin America thereby jeopardizing intercontinental banana production [4][5][6] . Management actions including crop rotation, ood fallowing, organic amendments, intercropping, molecular and biological control, etc have been applied to combat this disease but these measures provide short-term or little success under eld conditions that advocate for continuous exploitation of pugnacious methodologies that are oppugnant to the calamitous disease 6-10 .
Various research investigations of plant crude extracts revealed their inhibitory activities against phytopathogens that account for the presence of antimicrobial secondary metabolites as their compositional constituents. Additionally, these secondary metabolites e.g. terpenoids, alkaloids, tannins, saponins, phenylpropanoids, and avanoids, etc are vital materials in the manufacture of sundry fungicides and pesticides [11][12][13][14][15] . Secondary metabolites signify the adaptive potential of plants against biotic and abiotic stresses 16 . Secondary metabolites structure, optimized through evolution, interferes with microbes molecular targets hence acting as a mechanism for plant defense 17 . Phenolics are the profusely found secondary metabolites in plants 18 . Detection and identi cation of phenolics have now become an extensive research area because of the evidence that they have an indispensable role in the avoidance of the diseases that are linked to oxidative stress [19][20][21] . The plant phenolic compounds are studied as vital sources of novel antibiotics, insecticides, natural drugs, and herbicides 22,23 . Continuous exploitation of botanicals from various plants and their different parts would be productive in discovering innovatory, environmentally safe antimicrobials that can vanquish the complications of multi-drug resistance and bioaccumulation of pesticides.
Being used as folk medicines, gymnosperm botanicals have also been extensively studied for their antiin ammatory and antimicrobial potential in recent decades. The presence of diverse chemical constituents in these extracts is thought to be responsible for microbial growth inhibition [24][25][26][27] . The P. wallachiana (commonly called Biar or Blue Pine) is a large cone-bearing evergreen tree belonging to family Pinaceae of gymnosperms with a height up to 35-50m and a diameter of 1-1.5m, having downcurved branches with a straight trunk. Leaves are long (15-20cm), slender, in fascicles of 5, exible, the adaxial side having multiple bluish-white stomatal lines and abaxial side green 28, 29 . It is one of the principal conifers mostly growing in the upper region of mountains associated with other gymnosperms and is regarded as an important medicinal plant 30 . The majority of the research and pharmacognostic studies conducted on P. wallachiana strongly supported its antioxidant e cacies [31][32][33][34] and anticancerous potential of P. wallachiana needle extract 35 . Antibacterial activity of P. wallachiana essential oil against tested bacterial strains 36 and antifungal e cacy of its essential oil against Fusarium verticillioides 37 , antimicrobial activity of its hydroalcoholic extracts against tested bacterial strains and fungi 38 , antibacterial activity against Acinetobacter baumannii 29 put forward its antimicrobial potential. Phytochemical studies reported antioxidant activity of P. wallachiana extracts that accounts for the presence of plentiful avanoids and polyphenols in their phytochemical composition 32,39 . Phenolic compounds i.e. chlorogenic acid, catechins, ferulic acid, caffeic acid are wellknown toxic compounds that are much faster concentrated in resistant varieties after their infection by the pathogen 40 . Cell wall phenolics e.g. coumaric acid and trans ferulic acid play a crucial role during plant growth by defending it against stresses including infections and wounding etc 41 . The antiviral potential of catechins and (-)-epicatechin gallate against the in uenza virus had been noted. These polyphenols alter the membrane physical properties of the virus 42 . The antimicrobial potential of polyphenols e.g. catechin, gallic acid, ferulic acid, p-coumaric acid, quercitin, and rutin against Xylella fastidiosa had also been described earlier 43 . Similarly, antifungal activities of polyphenolics e.g. phenol, catechin, quercetin, ο-coumaric acid, gallic acid, pyrogallic acid, ρ-coumaric acid, ρ-hydroxy benzoic acid, protocatechuic, salicylic acid, coumarin, and cinnamic acid had been noted 44 . Moreover, powerful antimicrobial activities by polyphenol compounds including kaempferol, gallic acid quercetin, and ellagic acid had been reported 45 . Extracts abundant in antioxidants i.e. ascorbic acid, polyphenols, and avonoids are a source of cell damage and leaking of biomolecules from the impaired microbial membranes. The present study was designed to investigate the antifungal potential of Pinus wallachiana botanicals against Foc and evaluating its various fractions for the presence of some important polyphenols that might be bene cial for combating Fusarium wilt problem.

Materials And Methods
Acquisition, revival, and con rmation of fungal culture Fusarium oxysporum f. sp. cubense (Foc; TR4) was acquired from the Tissue culture department of National Agricultural Research Centre (NARC); Islamabad, the identity of which has been molecularly con rmed 46 . After the revival of Foc culture on potato dextrose agar (PDA), its morphology was examined; showing 3-5 hyaline, sickle-shaped septate, macroconidia pointed at both ends and borne on single phialides whereas microconidia were found to be mostly hyaline, kidney-shaped, aseptate produced on false heads.

Plant sample and extraction
A fresh leaf sample of P. wallachiana was collected from Ghora gali, Murree (altitude: 2291m, coordinates 33°54′15″N 73°23′25″E) and after its disinfection with 5% Clorox, it was shade dried for 30 days and then was mechanically toiled. Powdered leaf sample was stored in labeled plastic jars for the in vitro assays that were performed in the fungal pathology laboratory of NARC. The leaf powder was mixed with ethanol using Erlenmeyer asks, shaken at 60rpm (revolution per minute) for 48hours and after its ltration excess solvent was removed by the rotary evaporator 47 thereby dried extract was deposited in a glass vial 48 .

Fungicidal analysis
Two fold concentrations of the P. wallachiana leaf extract (1.25, 2.50, 5.0, 10, 20, and 40mg/mL) were amended in autoclaved PDA media for the determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) 49 . With the help of a plunger, 6mm wells were made in the center of poisoned plates and Foc plugs were aseptically placed followed by incubation (25±2˚C) and recording of MIC and MFC after a week's interval. Half minimal inhibitory concentration (IC 50 ) was also calculated using the regression equation 50 .

Effect on Foc biomass production
The liquid culture was used to evaluate the effect of the extract on the production of Foc biomass 51 .
Four treatments viz. control (no extract), IC50, MIC, MFC of the extract were separately dissolved in Potato Dextrose Broth (50mL). Each ask aseptically received 3-4 plugs of Foc and placed on a rotary shaker (90revolutions/min) and incubated (25±2˚C) for a month. Mycelia-containing asks were autoclaved and media was ltered and mycelia were dried overnight (40°C) after their washing with distilled water. Dry mycelia containing lter paper were then weighed and percent growth inhibition was calculated by equation (1) for each treatment as: P.I. = Dry weight of control-Dry weight of sample/Dry weight of control×100 (1) Where, P.I. = Percent inhibition Fractionation Liquid-liquid fractionation was performed for partitioning of P. wallachiana extract using a separating funnel 52 . The n-butanol, n-hexane, ethyl acetate, and dichloromethane were used as partitioning solvents. Fractionation was done in order of increasing polarity i.e. n-hexane>dichloromethane>ethyl acetate>nbutanol. The P. wallachiana extract was dissolved in water and sequential partitioning with n-hexane, dichloromethane, ethyl acetate, and n-butanol was done. Each fraction obtained was dried using a rotary evaporator and after calculation of its percentage yield using equation (2), stored in labeled glass vials.
Yield= weight of dried fraction/initial weight of extract × 100 (2) Antifungal assay of fractions Food poisoning assay Sterilized PDA plates poisoned with each fraction (10% conc.) and their 5% respective solvents that served as control were inoculated with Foc plugs (6mm) and incubated at 25±2˚C in ve replicates 53 .
When Foc mycelial growth completely covered all the control plates, radial mycelial growth was measured as the percent inhibition of Foc using equation (3). P.I. = Radial mycelial growth of control-Radial mycelial growth of treatment/ Radial mycelial growth of control×100 (3) Where, P.I. = Percent Inhibition Well diffusion assay Spore suspension (10 6 ) of Foc was spread on the entire surface of sterilized PDA as described earlier 54 . With the help of a cork borer, a hole with a diameter of 6mm was punched aseptically in the center of 9cm Petri plates (NEST, UK), and 100 µL from each fraction (10%) was introduced into the wells. Plates were incubated at 25±2˚C in ve replicates for all the treatments. Zone of inhibition (ZOI) started appearing after 3 days of incubation and was measured after one month.

Greenhouse experiment
Dwarf Cavendish banana plantlets (six weeks old) were acquired from a tissue culture laboratory, NARC. A double pot system (15cm×15cm×12cm) was used for banana plantation with a potting mixture of soil, sand, and peat moss in a 2:1:4 ratio. Millet grains colonized with Foc (50g) were packed in the middle of potting mix in each double pot system 55 to serve as inoculum. Treatments were applied as soil drenching 56 after banana plantlet sowing. Two concentrations of fractions (20mg/mL and 40mg/mL) and propiconazole (100µg/mL and 200µg/mL) along with their respective controls were used as soil drench treatments.
Three drenching's were applied during the greenhouse experiment and assessment of visual symptoms was done after each drenching. Evaluation of disease severity based on visual symptoms was measured 57 and Disease Severity Index (DSI) for each treatment was calculated.

HPLC analysis of fractions
Nine polyphenolic standards were used in HPLC analysis for their detection and quanti cation in P.
wallachiana fractions 58 . Fractions (1mg/mL concentration) were ltered with the help of a Membrane lter (0.45µm) and analyzed on Perkin Elmer HPLC system equipped with LC 295 UV/VIS detector, binary LC pump, and a reverse phase C18 column (4.6mm×250mm, 5µm). Solvent A (acetonitrile) and solvent B (distilled water/acetic acid, 99:1 v/v, pH 3.30±0.1) were used in combination to serve as a mobile phase. Linear gradient mobile phase with a ow rate of 1mL/min and 20µL injection volume of the sample was employed with detector setting at 285nm and 370nm for phenolics and avanoids respectively. Gallic acid, epicatechin, catechin, trans-ferulic acid, and trans-p-coumaric acid were used as phenolic standards

Fungicidal analysis
The MIC and MFC of P. wallachiana extract against Foc were determined to be 20mg/mL and 40mg/mL respectively while IC 50 was calculated to be 6.09mg/mL using regression equation (Table 1).

Greenhouse experiment of fractions
First severity scoring (based on a 1-5 scale) was performed after a month of 1st drenching. Highest disease severity index (DSI) value, calculated from the severity scores, was recorded for n-butanol fraction (40mg/mL) while n-hexane fraction (20mg/mL) along with dichloromethane fraction (20mg/mL) displayed lowest DSI. Second drenching was applied after recording rst severity scoring and second severity scoring was performed after two months of 2nd drenching. Maximum DSI i.e. 100% value was calculated by all solvent control treatments including fungicide (200µg/mL) and n-butanol fraction (20mg/mL). After second severity scoring third drenching was applied. Third severity scoring was performed after four months of 3rd drenching. Lowest DSI was noted for dichloromethane (20mg/mL) and hexane (40mg/mL) fractions with 60% values. Comparison of the DSI of different treatments, calculated at 3 different intervals revealed that progress of wilting was delayed in the case of dichloromethane (20mg/mL) and hexane (40mg/mL) fractions. Except for n-hexane, all the other fractions recorded maximum DSI in their higher concentration i.e. 40mg/mL (Table 5 & Supplementary  Table S5-S10). Same superscript letters within an individual severity scores column do no differ statistically and a common letter sharing between the treatments indicate non-signi cant difference. Disease severity index was calculated using formula. Seven replicates for each treatment.
Identi cation and quanti cation of polyphenolic compounds i.e. phenolic acids and avonoids were determined in the four fractions of P. wallachiana using HPLC analysis. Identi cation and quanti cation of phenolics (285nm) and avanoids (370nm) was according to retention time (RT) and peak spectral characteristics against those of standards. Detection of Polyphenolic compounds compared to standards and the overall polyphenolic content of P. wallachiana leaf extract varied in different fractions, as evident from the data (Table 6 & Supplementary Table 11S). The HPLC chromatograms of polyphenolic standards and two fractions of P. wallachiana i.e. ethyl acetate and n-butanol showed that all the polyphenolic compounds were detected in the n-butanol and ethyl acetate fractions except for rutin. Likewise, only quercitin and ferulic acid were detected in the n-hexane fraction while dichloromethane fraction detected all polyphenolic compounds except rutin, myrecitin and catechin ( Fig. 1 &  Supplementary Fig. S1). Highest gallic acid (11.57mg/g), catechin(33.44mg/g), epicatechin (16.74mg/g) and coumeric acid (4.33mg/g) were detected in n-butanol fraction whereas highest ferulic acid (2.84mg/g), myrecitin (2.15mg/g), quercitin (7.9mg/g) and kaempferol (7.81mg/g) were quanti ed in ethyl acetate fraction.
Maximum polyphenolic content, based on 9 polyphenol standards, were determined for n-butanol fraction of P. wallachiana (68.52mg/g of extract) followed by ethyl acetate fraction (43.90mg/g of extract) ( In the present investigation antimicrobial potential of Pinus walliachina; a gymnosperm was explored against one of the most devastating pathogen Fusarium oxysporum f. sp. cubense. Initial screening was carried out with P. walliachina leaf extracts for testing the antimicrobial potential. Results indicated that extract effectively inhibited the growth of Foc and based on these observations further experiments were initiated which included extraction of fractions using four solvents viz. hexane, dichloromethane, ethyl acetate and n-butanol and their potential to inhibit fungus in in vitro and green house. Both the assay results veri ed the effectiveness of P. walliachina and therefore, this study demonstrates rst report of antimicrobial activity of P. walliachina against Foc to the best of our knowledge. HPLC was also carried to further characterize all fractions and nine standards were used for the said purpose.
Leaf extract completely inhibited the mycelial growth of Foc and this observation was similar to the ones made previously where antifungal e cacy of extracts from distinct species associated with different families of gymnosperms was demonstrated 26 and also the e cacy of botanicals extracted from P.
walliachina exhibited prominent antifungal, antibacterial and insecticidal activities 31,37,38,66 . The four fractions of P. walliachina recorded signi cant percent inhibition and zone of inhibition against Foc in the poisoned food and well diffusion assays respectively. These results are in consistent with another study where fractions of P. walliachina crude leaf extract showed insecticidal (ethyl acetate) and antimicrobial (n-hexane) activities against Rhyzopertha dominica and Microsporum cannis respectively 67 . The nbutanol followed by dichloromethane fraction was found to most e cient treatments. However, the inhibitory potential of the four fractions was variable that might be due to different types of solvents used. It is reported that type of plant/plant part and type of extraction solvent are reason for the variation of phytochemical composition of various extracts 68 .
In the greenhouse assay the n-hexane fraction treatment with 40mg/mL and dichloromethane fraction treatment with 20mg/mL concentrations were found effective. Complete mycelial inhibition of Foc in the in vitro assay was observed in n-butanol fraction whereas in green house experiment same fraction (40mg/mL) recorded 100% DSI after one month of its very rst drenching. It was noticed that polar fractions with their higher concentrations recorded comparatively higher DSI values suggesting that with the high polarity of fraction its phytotoxicity to banana plantlets also increases. Polar fractions might have such phytochemicals that were not only detrimental to Foc but also had a phytotoxic effect on banana plantlets. A similar phytotoxicity phenomenon was described in an earlier study while working with different concentrations of chemical treatments (sterilant and fungicide) as soil drenching. All chemicals with 50 µg/mL concentration developed severe phytotoxicity symptoms while at lower concentrations none of the banana plantlets expressed phytotoxicity 56 . Similarly, in another study signi cant phytotoxicity of various fractions of P. wallachiana leaves at 500µg/mL were observed 67 . It can be concluded therefore that polar fractions should be used with comparatively lower concentrations i.e. less than 20mg/mL to decrease the DSI values.
The HPLC analysis of P. wallachiana fractions was done for the identi cation and quanti cation of polyphenolics using nine standards and it con rmed the presence of most of polyphenolic compounds in P. wallachiana fractions. All polyphenolic compounds except rutin were detected in the ethyl acetate and n-butanol fraction of P. wallachiana that commensurates with past study describing that all pine extracts contain a high number of polyphenols 69-71 . Dichloromethane fraction detected all polyphenolic compounds except rutin, myrecitin and catechin while n-hexane fraction only detected ferulic acid and quercitin. Epicatechin, gallic acid, coumeric acid, and catechin were recorded highest in n-butanol fraction while kaempferol, ferulic acid, quercitin and myrecitin were detected highest in ethyl acetate fraction. The highest polyphenolic content based on the 9 polyphenolic standards was quanti ed for P. wallachiana nbutanol fraction followed by ethyl acetate. An earlier study found quercetin as the most abundant avonol in n-butanol fraction (15.714%) of P. wallachiana methanol leaf extract using HPLC 72,73 . Similarly high amounts of polyphenolics mainly taxifolin and catechins were found to be the main reason for the antioxidant and biological activity of Pinus species 74 . Moreover, phenolics and sulfur present in the plant extract contributed to the cell death of Foc TR4 by inducing oxidative bursts, mitochondrial impairment, and depolarization of plasma membrane 75 . The production of phenolics in the resistant varieties of banana restricts pathogen to infected vessels due to ligni cations of obstructions resulted from initial pathogen-induced occlusion reaction 76 . There is evidence that trans-ferulic acid and pcoumaric acid signi cantly inhibiting the mycelial growth of Foc TR4 77 . The presence of the polyphenolic compounds quanti ed in the fractions of P. wallachiana, is the most probable reason for its mycelial inhibition activity against Foc.

Conclusion
This study exclusively evaluated P. wallachiana and its fractions e cacy against Foc and noted signi cant antifungal activity using in vitro and greenhouse assays, suggesting their potential role in the management of vascular wilt of bananas. It is established that polyphenolic compounds have potent e cacy against phytopathogens as we know this fact that phenolic compounds are active in plant defense response. HPLC analysis of P. wallachiana fractions revealed the presence of most of the compounds (based on 9 polyphenolic standards), with their maximum quanti cation in n-butanol and ethyl acetate fraction. The existence of such important polyphenols with known antimicrobial e cacy accounts for the antifungal activity of the P. wallachiana fractions against Foc that is contemporary scienti c information never reported prior. The research study strongly recommends P. wallachiana and its fractions be exploited further for the presence of valuable compounds that can make a breakthrough for control of Panama wilt disease soon.