Conditioning and Germination Control bio-assays of Quercus suber L. acorns under the Allopathic and Residual effects of Volatile Oils and Hydrolates from Aromatic Plants in Northeastern Algeria

 

Mohamed SEBTI¹*, Samir Benamirouche²

¹Laboratory of Environment, Biotechnology and Health,

Faculty of Nature and Life Sciences, University of Jijel, 18000 Jijel, Algeria.

²National Institute of Forest Research, Regional Station of Jijel, Algeria.

*Corresponding Author E-mail: sebtimoh@gmail.com

 

 

INTRODUCTION:

Cork oak (Quercus suber L.) is an endemic species of the Mediterranean basin, present in the western Mediterranean region for over 60 million years¹. A large proportion of the cork oak acorn’s collected during the ripening period (November-January) is used for the cultivation of nursery stock for reforestation to palliate the natural regeneration deficiency that has long been reported for the species.

 

After harvest, the acorns are stored in cold chamber until sowing in the nursery in the spring following their maturity. Indeed, to palliate the irregular periodicity of acorns production and to sow in the appropriate time, the safe storage of Q. suber acorns is unavoidable. The conditioning of these recalcitrant seeds that rapidly lose viability under ambient conditions remains, however, not fully resolved. In addition to the difficulty to maintain their high moisture content, fungus development, in particular Ciboria batschiana (Zopf) Buchwald (Ascomycetes, Sclerotiniaceae), and early germination during conditioning are from the other factors that limiting their long-term conditioning^2-4. Though the conditioning duration of these recalcitrant seeds has been significantly extended to up to two years², more research is needed to further address these limiting factors. This preliminary trial on new conditioning conditions for acorns could be a key element in the preservation and management of the genetic resource at the level of forest nurseries, especially if this resource is subject to natural disasters such as fires, drought, etc.

 

In studies which focused mainly on Sessile (Q. petraea M.) and Pedonculate (Q. rubor L.) oaks which consisted on a physical thermotherapy treatment in water followed by a chemical fungicide treatment prior to acorn conditioning; it has been demonstrated that this process doesn’t provide an effective control of Ciboria batshiana proliferation during acorn conditioning⁵. Apart from these studies, there are no additional reports related to the control of pathogens and early germination during acorn conditioning.

 

The development of plant pesticides made use of mainly secondary metabolites of plants, such as alkaloids, quinones, flavonoids, glycosides, saponins, tannins and terpenoids⁶, expected to play a crucial role in defence against pathogens and pests⁷. Biopesticides are used for their repellent, contact or fumigant effects, as volatile oils, macerates, decoctions and infusions. Of these phytochemicals, volatile oils obtained from different parts of aromatic and medicinal plants and their components which consisted on a complex mixture of esters, aldehydes, ketones, terpenes, and other low-molecular-weight volatile compounds⁸, have aroused the scientific community’s interest in recent decades for their phytosanitary activity⁹. Plant volatile oils recently have been used as a biological control for insects and pests through their usages as anti-fungal, anti-microbial, and in allelopathic potentialities for weed control as well as in seed protection¹⁰, but the mechanism of action is often nor entirely understood¹¹. In addition to volatile oils, plant distillation also generates a co-product known as hydrosol or hydrolate mainly used in food and cosmetic industries for their organoleptic and biological properties¹². For instance, due to potent antioxidant activity of grapes, their use in cosmetics is increasing day by day¹³. Since then, the use of volatile oils as alternative biopesticide has attracted considerable interest all over the world where several aromatic plant species have been studied for the usefulness of their volatile oils and hydrolates in human health and plant pest management. Accordingly, several papers and reviews have been published on the occurrence of antifungal compounds in plant and their potential use for human care and phytosanitary purposes¹⁴. To date, the phytosanitary role of number of plants and their phytochemicals in pest management is still being enthusiastically researched due to their less environmental and human undesirable side effects.  Many common Mediterranean aromatic plants belonging mostly to botanical families were studied for food tastes, pharmacopeia and phytosanitary purposes. Extensive data related to the chemical composition of volatile oils and hydrolates of several species and their potential uses is widely available, but to date studies have not looked at testing volatile oils and hydrolates obtained from the studied species during acorn conditioning. Moreover, the fast and not persistent performance of volatile oils related to their easy volatilization¹⁵ need to find a more effective application method to increase the duration of their effects required for safe long-term conditioning of acorns.

 

The present work deals with conditioning and control of early germination of Quercus suber acorns under the effects of plant volatile oils and hydrolates obtained by hydrodistillation from five common Mediterranean aromatic and medicinal plant species growing wild in Jijel in the north-eastern of Algeria including Calamintha hispidula Boissier and Reuter, Lavandula dentata L., Eucalyptus camaldulensis Dehnh, Pistacia lentiscus L. and Myrtus communis L. Therefore, this study was undertaken to demonstrate (i) the chemical profile of volatile oils and hydrolates of the five aromatic plants taken from Jijel (Algeria); (ii) their antifungal and allelopathic potential during acorns conditioning and iii) their residual effects under laboratory conditions with respect to germination and radical elongation. To the best of our knowledge, this conditioning and germination control technique was tested in this work for the first time. 

 

MATERIALS AND METHODS:

Plant material collect and preparation:

Areal parts of five Mediterranean shrubs evergreen species including Calamintha hispidula Bois. and Reut. (Lamiaceae), Lavandula dentata L. (Lamiaceae), Eucalyptus camaldulensis Dehnh. (Myrtaceae), Pistacia lentiscus L. (Anacardiaceae) and Myrtus communis L. (Myrtaceae) at vegetative or flowering stage, were collected from different locations from Jijel in the North-eastern of Algeria. C. hispidula were collected from El Anser (36° 47′ 52.08″ N; 6° 11′ 21.15″ E), L. dentata (36° 48′ 11.75″ N; 5° 45′ 09.04″ E.); E. camaldulensis (36° 48′ 14.45″ N; 5° 45′ 04.73″ E); P. lentiscus (36° 45′ 37.17″ N; 6° 16′ 45.59″ E) and M. communis (36° 45′ 37.34″ N; 6° 16′ 45.76″ E) were collected from Jijel. The samples were identified by Drs. De Belaire G. (University of Annaba) and Bouldjedri M. (University of Jijel) and confirmed by specimens (LDC13 ; LL1 ; ME148; AP1; MM1, respectively) deposited in the herbarium of the national park of Taza in Jijel.   

 

After harvest, the aerial part of the whole plant of C. hispidula, the branches of P. lentiscus, the leaves of  Myrtus communis and Eucalyptus camaldulensis and the flowering tops of L. dentata, were hand cleaned from impurities and shad dried at laboratory temperature for few days.

 

Cork oak acorn collect and preparation:

The acorns used in the experiment were provided by the forest conservation of Jijel. Acorns were extracted from a lot of acorns hand-collected in November 2017 from a seed source located in Kissir region in the northeastern of Algeria (36°79’ N, 5°66’ E) and stored in cold chamber maintained at 2-5°C. Acorns were then subjected to a vigorous cleaning conducted in two steps (water flotation followed by visual cleaning) to remove immature, dried, insect-infested and empty acorns. A lot of intact acorns were then obtained to perform the biological tests. 

 

Extraction and analyse of volatile oils and hydrolates:

Dried plant material of a given species was subjected to hydrodistillation for 2 h30- 3h, using Clevenger type apparatus appropriate for extracting volatile oils that are lighter than water^16,17. The hydrolates, designed as co-product of hydrodistillation were also collected during the distillation process. At the end of the distillation process, volatile oils and their corresponding hydrolates were put in hermetic glass bottles and kept refrigerated at 4°C until they were used.

 

The composition of the volatile oils was obtained using gas chromatography coupled to a mass spectrometry (GC-MS). The volatile oils were analyzed using GC/MS technique and several compounds belonging to different structural types were identified¹⁷. The chromatograph apparatus Schimadzu QP 2010 was coupled to an EI mode 70 eV quadropole mass spectrometer equipped with a SE 30 apolar capillary column. The split injector temperature was 250°C. The GC oven temperature was set at 40°C for 10 min, then 220°C at a rate of 5 C/min for 5 min. The transfer line being maintained at 250°C. Helium was used as a carrier gas at a constant flow of 2.0mL/min. Source and interface temperatures were set at 200°C and 250°C. The mass range was 40-450amu. Component identification for a given volatile oil were made by comparing mass spectra from the total ion chromatogram and retention indices using bank ESO 2000 computer database: library Nist 05. The amounts of the major compound in the hydrolates were simulated from those of the corresponding volatile oils.

 

Allelopathic bioassay:

The experiment was conducted following Araniti et al. ¹⁸, which was based on the procedure developed by Barney et al. (2005), with some modifications. Plastic boxes at the bottom of which was placed water-soaked cotton were prepared for the bioassay. Main component of cotton is cellulose which by it chemical composition and capillary structure can absorb and bind water molecules from atmosphere¹⁹. Afterwards, 50 acorns were placed on the water-soaked cotton. Each 10 acorns constitute a replication. Therefore, five replicates were performed for each treatment.

 

Application of the volatile oils and hydrolates:

For each plastic boxe, 1ml eppendorfs were attached to the inside of the cover of the boxe with a drawing pin (i.e. a concentration of 1ml/750cm³) and filled with volatile oils. The eppendrofs were kept open to ensure the action of the volatile compounds of the oils. The boxes were thereafter closed by their covers to prevent any volatile oil loss due to their inherent volatility. The hydrolates were directly sprayed on acorns in such a way to create a film. All the treatments including control were kept under conditioning conditions for a period of 30 days for fungi-proliferation and acorn pre-germination assessment. Plastic boxe without volatile oil or hydrolate was used as control.

 

Fungi-proliferation was expressed as the number of infested acorns with common symptoms; Infested acorns turn black and become spongy when pressed or cracked, revealing the discolored and decayed cotyledons. The number of infested acorns was monitored every three days until the end of the test predefined at 30 days. Infestation percentages were calculated based on the following equation: I % = nIA X 100/nTA

 

Where, nIA and nTA represent the number of infested acorns and the total number of acorns used for each treatment, respectively.  Germination defined as radical protrusion and elongation by at least 5mm was determined by counting germinated seeds every 3 days (72hours). Germination percentages were calculated on the 30th day as the ratio between the number of germinated acorn and the total number of acorns used in each treatment (volatile oils and hydrolates).

 

Residual effects of oils and hydrolates:

To assess the persistence of the effects of oils and hydrolates, an experiment was carried out in non allelopathic conditions. At the end of the allelopathic bio-assay, after 30 days, acorns previously treated by oils and hydrolates were transferred to plastic boxes free of oils and hydrolates an then incubated for 8 days under laboratory conditions. The residual effects of volatile oils and hydrolates were evaluated through measurement of germination and radical elongation. Percentage of germinated seeds was measured once at the 8th of incubation, whereas radical elongations were measured twice at the 1th (E1); the 4th and 8th (E2) days of incubation using a digital calliper.    

 

Experimental design and statistical analysis:

The experiment was conducted in a completely randomized design, with five replications per treatment. Statistical analyzes were performed using the Maxstat pro 3.6^© software and the means of germination and antifungal activity were compared by the Newman- Keuls test at 5% of error probability. A value of p ≤0.05 was considered to be statistically significant²⁰.

 

RESULT:

Chemical composition of volatile oils:

Table 1 list some results of the chemical composition of the five volatile oils extracted by hydrodistillation coupled with gas chromatography mass spectrometry (GC-MS) analysis.

 

Table 1. Major compounds of the volatile oils from the five studied species

Vo: Volatile oil ; Hyd : Hydrolate; RT: Residence time

 

GC-MS revealed that a total of 8; 8; 8; 22 and 7 different major compounds were identified in C. hispidula, L. dentata E. camaldulensis, P. lentiscus and M. communis volatile oils, respectively. These totals represented 89.55%; 100%; 100%; 82.35% and 91.74% of the total compounds of C. hispidula, L. dentata E. camaldulensis, P. lentiscus and M. communis volatile oils, respectively. 

 

As it can be seen in table 1, most of the identified compounds were monoterpenes (C10) which accounted for 88.27 % in the volatile oil of C. hispidula, 87.54% in M. communis, 98.68% in L. dentata, 43.58% in P. lentiscus and 57.95% in E. camaldulensis. Sesquiterpenes (C15) were highly present in E. camaldulensis (40.07%) and P. lentiscus (22.15%), weakly present in C. hispidula (1.28%) and totally absent in both M. communis and L. dentate oils. Diterpenes (C20) were only identified in P. lentiscus (15.54%) volatile oil.

 

The amounts of the major compounds of the hydrolates resulting from the simulation of their amounts in the respective volatile oils are given in the table 1 showing a height quantitative difference in the proportion of the major compounds in hydrolates from that in volatile oils. With values ranging from 0.79 to 1.66%, the concentrations of the major compounds in hydrolates were much lower than their amounts in the corresponding volatile oils.

 

 

Figure 1. Kinetic of acorn contamination C% (A; C) and acorn germination G% (B; D) with respect to the five volatile oils (VO) and their hydrolates (Hyd). (C.h.: Calamintha hispidula; L.d.: Lavandula dentate; E.c.: Eucalyptus camaldulensis; P.l.: Pistacia lentiscus; M.c.: Myrtus communis).

 

Effects of volatile oils and hydrolates:

The figures (1A, 1B, 1C and 1D) showed the evolution of contamination and germination of acorns under the effects of volatile oils and their hydrolates during the 30 days of the essay period. The fungistatic effect was much more interesting for volatile oils exhibiting 0 to 10% of contamination (Figure 1A) than for hydrolates exhibiting 10 to 80% of contamination (Figure 1C). Concerning germination, allelopathic effects were more pronounced for volatile oils where germination rates of 10 to 20% were registered (Figure 1B) as compared to hydrolates exhibiting germinations ranging from 50 to 90% (Figure 1D).

 

Volatile oil and hydrolate effects:

The antifungal activities of volatile oils extracted from the five species are presented in figure in figures 2 and 3, respectively.

 

According to our results, the highest antifungal activity was observed in the case of P. lentiscus and M. communis volatile oils where no contamination was registered during the 30 days of the bio-assay (Figure 2), which indicates the potent antifungal and fungistatic activity of these two oils. A contamination percentage of 10% succeeded by a fungistatic effect was registered in both oils from C. hispidula, L. dentata and E. camaldulensis. These oils prevent the spread of fungal spores from contaminated acorns to the other acorns.  In contrast, the control exhibited the weakest antifungal activity. The contamination percentage in the control increased from 30% on day 5 of exposure to 70% at the end of the bio-assay.

 

As can be shown in figure 3 both volatile oils showed high antigerminant activity compared with the control. The germination percentages registered at the end of the bioassay were 10% for acorns treated by M. communis oil and were 20% for acorns treated by hispidula, L. dentata, E. camaldulensis and P. lentiscus oils, whereas the highest germination percentage of 50% were registered in the control. Importantly, although the obtained results showed the effectiveness of the studied volatile oils in controlling fungi infestation and early germination of acorns, they also confirmed these limiting factors in cork oak acorn conditioning as it can be drawn from the results observed in the control treatment that has been the least effective. 

 

The antifungal and antigerminant activities of the hydrolates obtained from the five species are presented in figures 1 and 2, respectively. The obtained results show that most of the hydrolates do not have significant activity when compared to their corresponding volatile oils. The antifungal effects of C. hispidula and M. communis hydrolates were found to be the highest among all hydrolates tested (10% and 20% of contamination, respectively), L. dentata and E. camaldulensis hydrolates showed moderate antifungal effect with respectives contaminations of 30% and 40%, whereas P. lentiscus Hydrolate showed the weakest antifungal effects where 80 % of acorns were infested.

 

Overall, germination percentages recorded during conditioning for hydrolates were higher than those recorded for the oils and the control. In particular, P. lentiscus and M. communis hydrolates showed the highest germinations of about 80% and 90%, respectively. Acorns treated by the hydrolates from C. hispidula and E. camaldulensis showed 60% of germination, while an equal germination percentage of 50% was recorded in both L. dentata hydrolate and control.

 

Figure 2. Antifungal effects of the five volatile oils (VO) and their hydrolats (Hyd) on the final percent of contamination (C%) of acorn during storage. Bars with different letters (a, b, c, etc.) and (A, B, C, etc.) indicate significant differences at p < 0.05 level according to Newman-Keuls’s ranking test. (C.h.: Calamintha hispidula; L.d.: Lavandula dentate; E.c.: Eucalyptus camaldulensis; P.l.: Pistacia lentiscus; M.c.: Myrtus communis)

 

Figure 3. Effects of the five volatile oils (VO) and their hydrolats (Hyd) on the final percent of early germination (G%) of acorn during storage. Bars with different letters (a, b, c, etc.) and (A, B, C, etc.) indicate significant differences at p<0.05 level according to Newman-Keuls's ranking test. (C.h.: Calamintha hispidula; L.d.: Lavandula dentate; E.c.: Eucalyptus camaldulensis; P.l.: Pistacia lentiscus; M.c.: Myrtus communis).

 

Residual effects of volatile oils and hydrolates:

The results concerning the residual effects of volatile oils and hydrolates are presented respectively in figures 4 and 5, whereas the figure 6 presented the radicle elongation after 8 days of growing in in non allelopathic bio-assay. Overall, germination parameters (percentage and radicle elongation) were stimulated in post-conditioning bio-assay. After 8 days of incubation in non allelopathic conditions, germinations were promoted by 50% and 80% for oils and by 10% for both hydrolates.

 

Figure 4. Residual effects of volatile oils on the germination (G%) of acorns in non allelopathic bio-assay between the 30th and 38th day.  Bars with different letters (a, b, c, etc.) indicate significant differences at p<0.05 level according to Newman-Keuls's ranking test. (C.h.: Calamintha hispidula; L.d.: Lavandula dentate; E.c.: Eucalyptus camaldulensis; P.l.: Pistacia lentiscus; M.c.: Myrtus communis).

 

Figure 5. Residual effects of hydrolats on the germination (G%) of acorns  in non allelopathic bio-assay between the 30th and 38th day.  Bars with different letters (a, b, c, d) indiacted significant differencies at p<0.05 level according to Newman-Keuls's ranking test. (C.h.: Calamintha hispidula; L.d.: Lavandula dentate; E.c.: Eucalyptus camaldulensis; P.l.: Pistacia lentiscus; M.c.: Myrtus communis).

 

With the exception for the hydrolate of L. dentata for which germination of acorns seems to be prevented by the persistence of the phytotoxic effect during the conditioning period, though, without causing a marked reduction in root elongation at 8 days after growth in non allelopathic conditions.

 

On average, radicle elongation seems to be more stimulated by hydrolates (0.26mm) than by oils (0.16 mm). The lowest elongation (0.06mm) was observed with P. lentiscus oil, whereas the highest elongation (0.62mm) was observed with C. hispidula hydrolate.

 

Figure 6. Radicle elongation of acorns previously treated by volatile oils (VO) and their hydrolats (Hyd) after 8 days of growth in non allelopathic bio-assay. Bars with different letters (a, b, c and d) indicate significant differencies at p<0.05 level according to Newman-Keuls's ranking test. (C.h.: Calamintha hispidula; L.d.: Lavandula dentate; E.c.: Eucalyptus camaldulensis; P.l.: Pistacia lentiscus; M.c.: Myrtus communis)

 

DISCUSSION:

The present work studies for the first time the effectiveness of secondary plant metabolites during cork oak acorn conditioning, already limited by fungi proliferation and acorn pre-sprouting. Accordingly, volatile oils and their hydrolates were extracted from five Mediterranean evergreen shrub species grown wild in north east of Algeria, were subjected to a GC-MS to identify their major compounds and were then used to treat cork oak acorns maintained in cold room for 30 days during which fungi-infested and germinated acorns were recorded. At the end of this period, acorns were transferred to plastic bottles free of oils and hydrolates and kept under laboratory condition for persistence effects assessment with respect to germination and radicle elongation.

 

Chemical composition of the extracted volatile oils done using gas chromatography-mass spectrometry (GC-MS) showed difference in quantitative and qualitative composition among the studied species (Table 1). Overall, monoterpenes were found to be the dominant compound followed by sesquiterpenes and diterpenes, respectively. Monoterpenes were the highest proportionally in L.dentata (98.68%), C. hispidula (88.27%), M. communis (87.54%), E. camaldulensis (57.95%) and P. lentiscus (43.58%),  sesquiterpenes showed relevant amounts in E. camaldulensis and P. lentiscus oils (40.07 and 22.15%, respectively), whilst diterpenes were only present in P. lentiscus oil (15.54%). These findings are in agreement with Kordali et al.²¹, who reported that volatile oils of fruits of four M. communis genotypes from Turkey were characterized by high amounts of monoterpenes (73.02 - 83.83%). Some compounds were highly present in the studied oils such as α-pinene (40.22%) in M. communis, β-myrcene (24.75%) in P. lentiscus, isomenthone (36.17%) and pulegone (39.51%) in C. hispidula, spathulenol (40.07%) and quercivorol (31.64) in E. camaldulensis and thymol (22.65%) in L. dentata. It is also interesting to note the relevant amounts of 1-p-menthol (9.12%) in E. camaldulensis, α-terpineol (9.84%) and linalool (7.19%) in L.dentata. Other compounds were present in small proportions; piperitone oxide (4.90%) and d-limonene (3.58%) in C. hispidula, trans-pinocarvacrol (4.44%) and γ-terpinene (3.08), ο-cymene (4.56%) in E. camaldulensis, β-pinene (3.67%) and α-pinene (3.47%) in P. lentiscus, d-limonene (5.29%) and α-terpineol (4.05%) in M. communis. Some compounds were simultaneously present in different species such as 1-8 cineol in both L. dentata (47.55%) and M. communis (33.36 %), d-limonene in both M. communis (5.29%), C. hispidula (3.58%) and P. lentiscus (2.36%) and α-pinene in both M. communis (40.22%) and P. lentiscus (3.47%).

 

The current study joint the wide qualitative and quantitative variability in volatile oils composition reported for many species, even when belonging to the same family as can be easily seen here for Myrtaceae (E. camaldulenis vs. M. communis) and Lamiaceae (C. hispidula vs. L. dentata). The eucalyptol also reported as 1-8 cineol, which is widely distributed in many plants²², was found in both L. dentata and M. communis with respective proportions of 47.55% and 33.36% of the fraction of oils analyzed. Several studies have reported high proportions of eucalyptol in L. dentata volatile oils, even when collected from different countries. The amount of eucalyptol was 63.5% in Tunisian L. dentata sample²³; 63% in Brazilian sample²⁴, 61.36% in Moroccan sample²⁵ and 34.33% in Mexican sample²⁶. The sesquiterpene spathulenol as the major constituent of E. camaldulensis volatile oil in the present study (40.07%) was higher than that of 22.05% reported by  Nait Achour et al.²⁷, for E. camaldulensis grown in Tizi Ouzou (North central of Algeria). The amounts of pulegone and piperitone oxide in C. hispidula oil found in the current study (39.51 and 4.90%, respectively) were different from those (24.72 and 33.95%, respectively) previously reported by Sebti et al.²⁸. Although camphor was completely absent in the L. dentata volatile oil characterized in the present study, it was highly present in oil extracted from L. dentata grown in northern Tunisia²⁹ and Morocco³⁰ with respective amounts of 35 and 64.43%. Gakuubi et al.³¹, reported that Kenyan E. camaldulensis oil was dominated by eucalyptol (16.20%), α-pinene (15.60%), α-phellandrene (10.0%), and p-cymene (8.10%), while in the present study E. camaldulensis oil was dominated by spathulenol (40.07%), quercivorol (31.64%) and 1-pmenthol (9.12%).

 

P. lentiscus volatile oil from Algeria is characterized by the presence of o-cymene (22.94%), menthone (12.09%), β-pinene (10.35%), α-pinene (9.41%), α-farnesene (9.22%), β-myrcene (7.69%), trans-pinocarveol (6.86%), limonene (6.34%), pulegone (5.29%) and verbenone (4.45%)³². For Djenane et al.³³ and Imelouane et al.³⁴, the volatile oil of P. lentiscus is constituted mainly of myrcene (15.18%) and 1,8-cineole (15.02%). α-pinene and β-pinene are known as compounds with antimicrobial potential. However, the antifungal activity of volatile oil of P. lentiscus is due to the α-pinene which constitutes a considerable amount of this oil.

 

These oil chemoptype disparities and many others mentioned in the literature such as those summarized for M. communis³⁵ and P. lentiscus³⁶ might be due to several factors, including the extraction process, isolation conditions (temperature, time, solvant, etc.), geographical origin and phenological stage of the plant source.

 

The results of the in vitro study showed that the five volatile oils were effective against fungi infestation already observed during cork oak acorn conditioning² and that the P. lentiscus and M. communis volatile oils were the most effective.  Fungi infestation was totally inhibited by application of P. lentiscus and M. communis volatile oils, acorns treated by L. dentata, C. hispidula and E. camaldulensis showed a fungal activity affecting 10 % of the seeds which, however, stabilised at this rate until the end of the bio-assay indicating a fungistatic effect, while the control free of oils showed the highest infestation of about 70%. Although difficult to identify the compound responsible for the antifungal activity as long as the oils have been used in their full composition, i.e. without isolation of the different active compounds, this antifungal activity might be due, in part, to the terpene compounds identified in the various species as it has been observed for antifungal activity of many monoterpenes and sesquiterpenes against various phytopathogenic fungi³⁷, although the effects of other minor components should not be overlooked³⁸. The identified compounds may act individually or in synergy as allelochemicals^39,40. For instance, thymol (22.65%) and linalool (7.19 %) present in L. dentata oil are known to have potent antifungal activity, as previously reported for thymol against the fruits and vegetable pathogens, and for linalool against Candida species⁴¹. In the same line, Boubaker et al.³⁰ revealed that L. dentata volatile oil (Eucalyptol, 64.43%) has been shown to inhibit the spore germination of the main post-harvest pathogens in citrus. Furthermore, an inhibitory effect at minimal concentrations of Chilean cultivated L. dentata oil (60% eucalyptol) against Candida albicans in antibiofilm assay was reported⁴². More recently, Yilmaz et al.⁴³ reported the high inhibitory effect of carvacrol as the major component of O. vulgare against Clavibacter michiganensis subsp. michiganensis infected tomato seeds. E. camaldulenis oils from various countries were shown highly effective against various Fusarium species³¹.

 

Apart the antifungal effect, the oils also showed an inhibiting effect on the germination of acorns. Among the five volatile oils tested, M. communis oil had the highest inhibitory effect on acorn germination. Minimum of germination during conditioning (10%) was shown by M. communis volatile oil (α-pinene 40.22%,  eucalyptol 33.36%), followed by an equal germination percentage of 20% shown by the four oils of C. hispidula isomenthone (36.17% pulegone 39.51%), L. dentata (eucalyptol 47.55%, thymol 22.65%), E. camaldulensis (quercivorol 31.64%, spathulenol 40.07%) and P. lentiscus (β-Myrcene, 24.75%), while the maximum germination percentage (50%) was shown by the control. Similar inhibitory effects of volatile oil on seed germination have been widely reported in literature particularly in the context of the study of the herbicidal effect against weeds as a safe alternative to synthetic pesticides^44-47. Bachheti et al.⁴⁸ presented terpenoids as one of the major allelochemicals present in plants which show inhibitory effects on germination and plant growth. Among several compounds, their review referenced the oxygenated monoterpenes 1,8-cineol, camphor, carvacrol, α-pinene and limonene as volatile allelochemicals with inhibitory effects. In earlier study. The results from this study suggest that both citronellol and linalool possess strong phytotoxic potential and can thus serve as lead molecules for the synthesis of bioherbicides.  The herbicidal effect of volatile oils obtained from four M. communis genotype from Turkey (1,8-Cineole 29.20-31.40, linalool 15.67 -19.13, α-terpineol 8.40-18.43, α-pinene 6.04-20.71 %) against some common weeds: Amaranthus retroflexus L., Chenopodium album L., Cirsium arvense (L.) Scop., Lactuca serriola L. and Rumex crispus was evaluted²¹. Their study showed complete or partial inhibition of seed germinations and seedling growths of the target plants which they attributed to the high contents of oxygenated monoterpenes, representing 73.02– 83.83% of the oils. In the same way, volatile oil of T. articulata (α-Pinene 56.21, β-myrcene 3.08, 1,8- cineole 9.91%) completely inhibited the seed germination of S. arvensis L. at high concentration (4μl/ml), while at low doses (1 and 2 μl/ml), it delayed the germination and reduced the seedling growth of both weeds S. arvensis L.  and Phalaris canariensis L.⁴⁹. Pesticidal testing of Atriplex cana volatile oil (dibutyl phthalate 21.79, eucalyptol 20.14 and myrtenyl acetate 15.56 %) strongly inhibited germination and growth of A. retroflexus, Medicago sativa L., P. annua and Echinochloa crusgalli ⁵⁰. Volatile oil of Silver Fir (Abies alba Mill.) needle extract (Limonene 15.99, α-pinene 11.87ng/mL) significantly delayed and reduced seed germination of the three weeds C. canadensis, C. album and A. retrofllexus⁵¹. Likewise, Mirmostafaee et al.⁵² reported that compounds such as borneol, eucalyptol, limonene, α and β-pinene, carvacrol, and camphene were the dominant compounds in volatile oils with severe inhibitory effects on lettuce germination, seedling growth and vigor. Being the dominant constituents in Serphidium kaschgaricu oil, eucalyptol and camphor and their mixture exhibited the strongest phytotoxic activity against A. retroflexus and P. annua³⁹. Accordingly, the authors suggested that less abundant compounds in the volatile oil of Serphidium kaschgaricu might contribute significantly to the oils’ activity. Pulegone and isomenthone  as the dominant compounds in  volatile oils of C. nepeta and M. pulegium, and also dominant in C. hispidula (pulegone 39.51%, isomenthone 36.17 %) in our study, were shown to exert strong inhibitory effects on germination and root elongation of Lactuca sativa¹⁸ and on germination of P. oleracea⁵³, respectively.

 

Also, the monoterpenes above mentioned (1,8-cineole, thymol, linalool, α-pinene, β-myrcene, limonene, pulegone, isomenthone and carvacrol) and recognized as the most active compounds in several works, were in general highly present in the oils characterized in the present study. Therefore, the bioactivity (antifungal and antigerminant activities) of the volatile oils here observed might be reasonably attributed to these constituents. On the other hand, the overall results of this study showed that hydrolates, designed as a co-product of hydrodistillation, were less effective than the volatile oils against fungi infestation and acorn germination during conditioning.  The highest antifungal activity was exhibited by the hydrolate from C. hispidula (10%), while hydrolate from P. lentiscus displayed the weakest activity (80%).  Likewise, germinations during conditioning were in all (60- 90%) higher as compared with the control (50%).This low bioactivity can be attributed to the low concentration of major compounds (0.86–1.66%) in comparison to that in the corresponding volatile oils. Indeed, it is known that the difference in the composition of a hydrolate from that of an volatile oil is mainly quantitative.  

 

Furthermore, in non allelopathic conditions, almost all studied volatile oils and hydrolates were shown to stimulate acorn’s germination and radicle elongation compared to the control. In the post-allelopathic germination bio-assay, germination was promoted up to 30% and more for acorns previously treated by volatile oils and by 10% for acorns previously treated by hydrolates, except for L. dentata hydrolate where no germination was observed. The P. lentiscus and E. camaldulensis oils showed the highest post-allelopathic germination increase of about 80%. However, the hydrolates were shown to stimulate radicle elongation more than volatile oils with average elongations of 0.26 and 0.16mm, respectively. In addition to the protective and disinfecting effects, we can suggest that volatile oils and hydrolates herein studied had no adverse effect on acorn viability since the high germinations registered in non allelopathic conditions. Therefore, and adding the fact that volatile oils were herein shown more effective than their hydrolates, we can suggest that the studied oils can be advantageously used as a safe alternative to synthetic fungicides for the control of fungi proliferation and early germination during cork oak acorn conditioning.

 

Finally, the results of these new conditions for the conservation of healthy acorns would certainly encourage the development of new techniques for the conservation of agricultural and food seeds through the formulation of new products, both biocides and which condition germination.

 

CONCLUSION:

The present study was undertaken to explore the chemical composition of volatile oils and their hydrolates obtained by hydrodistillation from five plant species growing wild in Jijel in the north-eastern of Algeria and to verify for the first time their antifungal, allelopathic and residual effects during the conditioning of cork oak acorns.  Chemically, the volatile oils of the studied species are dominated by oxygenated monoterpenes such as 1,8-cineole, thymol, linalool, α-pinene, β-myrcene, limonene, pulegone, isomenthone and carvacrol with recognized antifungal action and that are most likely responsible for the observed results. Moreover, given that volatile oils  have exhibited more antifungal  and allelopathic effects than hydrolates, we can suggested that volatile oils from both studied aromatic plant species are promising candidates in the search for new antifungal agents enabling the control of fungi-proliferation during cork oak acorn conditioning. Thus, the results presented here are promising and support the continuation of the search in this approach. Further studies to elucidate the possible compound(s) involved in the observed activities, the mechanisms of action, the durability of their action for a long-term conditioning and the most effective way for large-scale application will be undertaken.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGMENTS:

The authors gratefully acknowledge all staff of the nursery of the forest conservation of Jijel for providing acorns and an adequate environment in which this research was conducted. Likewise the authors are grateful to Dr. Mohamed Bouldjedri Lecturer at the Mohammed Seddik Benyahia University of Jijel for species identification and Mr Desdous Abderrachid for GC-MS analysis.

 

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Received on 17.10.2022                    Modified on 08.05.2023

Accepted on 25.09.2023                   ©AJRC All right reserved