Control of Lavandula Dentata Root Rot with Leaves Extracts of Lawsonia Inermis and Eucalyptus Camaldulensis

INTRODUCTION

The intensive and indiscriminate use of fungicides in agriculture has caused many problems to the environment such as water, soil, animals and food contamination, poisoning of farmers elimination of non target organisms and selection of phytopathogens, pest and weed insensitive  to certain active ingredients( Strangarlin et al., 1999).To minimize the negative effects of fungicides alternative developed  methods to  control  plant diseases are being used. These methods include the biological control, the induction of resistance and the use of natural products with induction of resistance and or with direct antimicrobial activities (Schwan and Strangarlin, 2005).

The biological control is defined as the use of antagonistic organisms for the control of microorganisms, reducing the amount of inoculum that determines the extent of disease (Cook and Baker, 1983).The induction of resistance promotes the activation of the latent plant defense systems, which manifests itself when it comes into contact with a biotic (Felipini and Di Piero, 2009) on apple and (Strangarlin et al., 2010) on soybean or abiotic elicitor carre  and ( Barretti et al., 2010) on tomato. The expression resistance of induction both can be used to denote local protection, this is, the induction of a resistance only in the tissues in which it was applied the treatment with the inducing agent, also can indicate a systemic resistance that manifest far from the tissue where the elicitor was applied ( Moraes, 1992).

Henna (Lawsonia inermis) belongs to family Lythraceae  is small shrub cultivated in many tropical countries and warm temperate reign. It has been used  for  coloring  palms of hands, soles of feed an finger nails and also for personal adornment (Bhuvane and Kuruvilia, 2002). Lawsonia inermis plant constituent are made up tannic acid, mucilage and gallic acid, but the main bioactive constituent is 2-hyrooxynaphthoquinon (lawsone).(Singh and Singh, 2001) powdered leaves of this plant in paste form have been used as cosmetic and as remedy in skin diseases and used as  external application in headache. Lawsonia inermis aqueous extract has been used as natural fungicide (Dahankhar et al., 2000). Also,  Lawsonia inermis aqueous extract has  a high antimicrobial activity against potato dry rot disease caused by Fusarium solani (Bhardwaj, 2012). Spraying Lawsonia inermis aqueous extract is the safe method to control powdery mildew disease of Zinnia elegans (Hegazi and  El- Kot, 2010).

Some components in Lawsonia inermis leaves  according to (Duke , 1992)

Camphor (Eucalyptus camaldulensis)  belongs to family  Myrtaceae, is one of  the reputed fast growing  trees of the world. It^,s oil is acrid, bitter, astringent and insect repellent,( Babu et al., 2008).

Some components in Eucalyptus camaldulensis leaves according to (Duke,  1992)

lavender (Lavandula dentata) belongs to family Lamiaceae. Lavandula dentata is one of the most useful medicinal and aromatic  plants. Commercially,  it is an important source of essential oil that  is widely used in fragrance industry including soaps, colognes,  perfumes, skin lotions and other cosmetics (Paul et al., 2004). In food manufacturing, lavender essential oil is employed in flavoring beverages, ice –cream, candy, baked goods and chewing gum (Kim and Lee, 2002) . Recently, aromatherapy is becoming increasingly popular and Lavandula dentata is used  in aromatherapy as a relaxant (Lis –Balchin  and Hart, 1999).Several  therapeutic effects of  Lavandula dentata, such as sedative, antiviral and antimicrobial activities have been reported (Gamez et al., 1990 ; Buchbauer et al., 1991).

Lavandula dentata essential oil  are advocated for their use as antibacterial agent in both early and modern aromatherapy texts (Lawless,1992; Gattefosse,1995). Lavandula dentata is exposed to many diseases and root rot is one of the Botryodiplodia theobromae (Pat.) Griff. and Maubl. (Syn: Lasiodiplidia theobromae Pat.) and its asexual state, Botryosphaeria rhodina (Berk and M.A. Curtis) Arx are fungal pathogens of great economic importance.It is a cosmopolitan fungus causing both field and storage diseases on more than 280 plant species including crops, fruits, and cash fruit trees .B. theobromae is an opportunistic plant pathogen that causes different types of plant diseases within tropical and subtropical regions ( Faber et al., 2007). It has a wide host range estimated to be more than 280 plant species (Domsch et al., 2007;Khanzada et al., 2006; Sutton 1980) although with varied pathological effects on its hosts. The fungus is known to cause tuber rots in yam, root rot in cassava, collar rot in peanuts, crown rot in banana, stem end rot in mango fruits, stem rot in pawpaw and leaf spot in citrus (Sangeetha et al., 2011; Rossel et al., 2008; Khanzada et al., 2004). Rots caused by the fungus, particularly in the root and tuber crops often occur underground and so diagnosis of the disease is usually delayed or under repaired. Moreover, the wider host range (Crammer, 1979) and the host non- specificity of B. theobromae makes control and management of the disease very difficult.

Nawadays synthetic fungicides are used as primary tools for the control of plant diseases. However, the alternative control methods are needed because of the negative public perceptions about using synthetic chemicals, resistance to fungicide among fungal pathogens, and high development cost of new chemicals. The uses of plant-derived products as disease control agents have been studied, since they tend to have low mammalian toxicity, less environmental effects and wide public acceptance (Lee et al., 2007; Katooli et al., 2011).             

The aim of this research was to examin the antifungal activity of aqueous extracts of Lawsonia inermis and Eucalyptus camaldulensis leaves  separately to control of Lavandula dentata plants root rot disease .

MATERIALS AND METHODS

           This experiment was conducted at the Experimental Field of the Medicinal and Aromatic Plants Department at Dokky, Giza, Egypt, in two successive seasons 2012 and 2013.

1. Preparation of Plant extracts

          Lawsonia inermis and Eucalyptus camaldulensis aqueous extracts were  prepared by soaking (100g) of  powdered  dried leaves in tap water  for 24 hours  and then they were filtered. Each extract was concentrated at  (50 and 100g/L) (Babu et al., 2008).Lavandula dentata plants were sprayed four times  with aqueous extracts of  Lawsonia inermis and Eucalyptus camaldulensis separately. The first spray was conducted after planting, the second was done three weeks there after, the third was applied after the 1^st cut and the fourth was added three weeks after the third one.

2.Experimental procedure

2.1.Isolation and identification of causal pathogen (Botryodiplodia  theobromae )         

Naturally  infected plants showed root rot disease  symptoms, collected from El –Ekhlas  Farm  at Giza ,were  cut into pieces 5mm long .These  pieces were surface  sterilized for 3min. with 3% sodium hypochlorite  and rinsed in 4 successive changes of sterile distilled water then left to dry on Whatman No. 1 filter paper .The surface sterilized pieces were transformed to potato dextrose agar (PDA) medium in petri  dishes  and incubated at  27± 2ºC  for 5 days . Pure cultures, made by single spore technique, was identified using cultural, morphological and microscopical characters  according to (Barnett and Hunter, 1981).

2.2.Soil infestation with the pathogenic fungus

Isolatedpathogen Botryodiplodia  theobromae  was grown on sand –corn medium(1:1w:w and 40% water ) for 15 days at 25± 2ºC.Pots (30cm diameter) containing sterilized sandy loam soil were artificially infested separately with prepared fungal inoculum at the rate 3% of soil weight .The inoculum was mixed thoroughly with the upper layer of the soil  then irrigated every other day and left for 7 days to ensure  the distribution of the inoculum. Each treatment was replicated three times every replicate consisted of nine pots (1 plant / pot)  and nine ones left without infestation to serve as control. Root rot percentage was recorded 45 days after planting.

2.3 Source of cutting   

Lavandula dentatacutting (15-20) cm in height were obtained from El- Ekhlas Farm at Giza and planted in plastic pots (30cm diameter) on  25^th  February, 2012 and 2013  in the first and second seasons, respectively.

3. Experiment layout

The experiment layout was designed in complete randomized blocks included five treatments each treatment was replicated three times and every replicate consisted of nine pots (1plant /pot), the recorded data were statistically analyzed according to Snedecor and Conchran (1968), using L.S.D at 5% .

4. Chemical fertilization

The sources of chemical fertilizers (NPK) were ammonium sulphate  (20.6%N), calcium superphosphate (15.5% P₂O₅) and potassium sulphate (48%K₂O). (NPK) fertilizers  were added at  the recommended level in five doses, the 1^st was  for all phosphorous amount which was added during soil preparation, the rest (NK) were applied in two equal doses for each cut, on 8 ^th April  and  10 ^th May  for the1^stcut  and  on 10 ^th June (after the1^st cut)  and 9 ^th July for the 2^ed one in the two seasons. The plants were harvested  twice, the first cut was conducted on 7^th June  and the second one  on  8 ^th August  in both  seasons.

5.Treatments

1.Control (Botryodiplodia  theobromae fungus all plants died and were not subject to statistical analysis ) .

2.Botryodiplodia  theobromae fungus+ Lawsonia inermis aqueous extract at (50g/L).

3.Botryodiplodia theobromae fungus +Eucalyptus camaldulensis aqueous extract at (50g/L).

4.Botryodiplodia theobromae fungus + Lawsonia  inermis aqueous extract at (100g/L).

5.Botryodiplodia theobromae fungus +Eucalyptus camaldulensis aqueous extract at (100g/L).

6.Data recorded

    The following data were recorded

1. Identification of causal pathogen (Botryodiplodia  theobromae )

2.Percentage  of root rot.

3.Plant height and number of branches /plant.

4.Herb fresh and dry weights (g/plant).

5.Volatile oil percentage in fresh herb according to (British Pharmacopeia, ,1963).

7.Volatile oil component

Sample taken from the oil obtained in the first cut of the first season were analyzed using gas liquid chromatography (GLC), to determine their main constituents. The use of GLC in the quantitative determinations was performed using the methods described by (Bunzen et al., 1969;  Hoftman, 1967).

RESULTS AND DISCUSSION

1. Identification of causal pathogen

Associated with root rot symptoms was a fungus that had the following morphology: solitary pycnidia, pyriform, black, 150–175 X190–210 mm, glabrous, with an apical ostiole, stromatic wall, composed of several layers of dark brown, thick-walled cells; conidia ellipsoidal, one-celled, hyaline when immature becoming dark brown, striate and didymospore with age ,22.0–28.5 X12.5–14.0 mm. The fungus fits the description of Botryodiplodia theobromae (Pat.) Griffon  & Maubl. (Punithalingam, 1976 ; Barnett and Hunter, 1981).

2. Percentage  of root rot

Aqueous extracts of Lawsonia  inermis and Eucalyptus camaldulensis were tested for their antifungal ability to control root rot disease of Lavandula dentata caused by the pathogenic fungus  Botryodiplodia theobromae . Data in Table (1) clearly emphasized that, Lawsonia inermis and Eucalyptus camaldulensis aqueous extracts separately control  root rot disease . It is obvious that resistance agents significantly decreased  the incidence of root rot disease, Lavandula dentata plants were protected by aqueous extracts of Lawsonia  inermis and Eucalyptus camaldulensis separately. The most effective treatment was aqueous extract of Lawsonia inermis at 100g / L in both seasons. This result may be due to medicinal and aromatic plants represent a rich source of antimicrobial agents ( Mahesh and Satish, 2008). Plants generally produce many secondary metabolites, fungicides and many pharmaceutical agents used in traditional medicine (Ibrahim, 1997; Ogundipe et al., 1998).Medicinal and aromatic plants  are the sources of natural fungicides that make excellent  leads for new fungicides development (Arokiyara et al., 2008; Brindha et al., 2009). Also Medicinal and aromatic plants have limitless ability to synthesize aromatic secondary metabolites, most of which are phenols or their oxygen subatituted  derivatives (Geissman, 1963). Important subciasses in this group of compounds include phenols, phenolic acids, quinones, flavones, flavonoids, tannins, coumarins and essential oils. These groups of compounds show antimicrobial  effect and serves as plant defense mechanisms against pathogenic microorganisms. Simple phenols and phenolic acid are bioactive phytochemicals consisting a single substituted phenolic ring. Phenolic toxicity to microorganisms is due to the sites and number of hydroxyl groups present in the phenolic  compound( Scalbert, 1991;  Ursand Dunteavy, 1975).Quinones are  characteristically highly reactive, colored compounds. These results were in accordance to ( Bambawale et al., 1995) on cotton found that,  Lawsonia inermis  aqueous extract inhibited spore germination and mycelial growth of Myrothecium roridum. Also, (Bakeer et al., 2005) on Pelargonium graveolens reported that, garlic (Allium sativum) and onion (Allium cepa) extracts significantly decreased the incidence of Pelargonium graveolens root rot disease. (Sharma et al., 2010) on kinnow fruits observed that, Allium sativum aqueous extract inhibited 100%of mycelial growth of Botryodiplodia  theobromae followed by aqueous extract of Lawsonia inermis which reduced  73.64% of rot incidence.   

Table 1. Effect of Lawsonia inermis and Eucalyptus camaldulensis leaves  extracts on percentage of  root rot               of Lavandula dentata plants during 2012 and 2013 seasons                                                                                                                                 

The same results were obtained by  (Hegazi and El-Kot, 2010) found that, spraying Zinnia elegans plants with Lawsonia inermis aqueous extract inhabited powdery mildew caused by Erysiphe cichoracearum fungus. In the same way, (Tariqu et al., 2010) on Gladiolus grandiflorus stated that, spraying both  aqueous extracts of Allium sativum and Allium cepa reduced incidence of corm rot disease caused by Fusarium oxysporium fungus.

3.Vegetative growth

3.1.Plant height and number of branches/ plant

 Data in Table (2) indicated that, treatment inoculated with Botryodiplodia  theobromae fungus alone  all plants died  (control), also both aqueous extracts of Lawsonia inermis  and Eucalyptus camaldulensis had a significant increased in plant height and number of branches / plant in the two seasons. The tallest  plants in the first and second seasons at the two cuts were recorded by the  plants were treated with aqueous extract of Lawsonia inermis  and Eucalyptus camaldulensis  separately at 100g/L which gave (36.36, 34.77cm) and (29.16, 27.36 cm) at first and second cuts in the first season, respectively. The same trend was observed in the second season giving (32.50, 31.77 cm) and (26.97,25.30 cm). The shortest plants in the two cuts of the first and second  season were treated with aqueous extract of Eucalyptus camaldulensis at 50 g / L as shown in Table (2).The same trend was observed in the case of number of branches. The application of Lawsonia inermis aqueous extract also had  a significant effect on number of branches/plant, the highest number of branches was recorded when plants were sprayed with Lawsonia inermis aqueous extract at 100g/L giving 9.35 and 10.07 at first and second cuts, respectively, while in the second season the recorded data were 12.01 and 13.15 at first and second cuts, respectively. The lowest values were recorded when the plants were sprayed with Eucalyptus camaldulensis aqueous extract at 50g/L in the first and second cuts at the first season giving (7.46 and 8.90).The same trend was observed in the second season giving (9.60, 10.96) at the first and second cuts.

These results may be due to Lawsonia inermis aqueous extract contains lawsone (C₁₀H₆O₃),the active

ingrediend and naturally occurring  naphthoquinone (Habbal et al., 2007) and the presence of eucalyplol in Eucalyptus camaldulensis  aqueous extract (Babu et al., 2008).These results are in agreement with that obtained by (Tariqu et al., 2010) on Gladiolus grandiflorus.

3.2.Herb fresh and dry weights (g/ plant)

         Data in Table (3) revealed that, treatment applied with the tested fungus alone all plants died (control).  Spraying   aqueous extracts of Lawsonia inermis and Eucalyptus camaldulensis separately had a significant effect on   herb fresh and dry weights in most cases in the two seasons. The best results were obtained from Lawsonia inermis aqueous extract. The highest fresh and dry weights /plant were recorded when Lavandula dentata   plants were sprayed with Lawsonia inermis aqueous extract at 100g/L giving 37.15, 39.31 g /plant and 41.43, 48.44 g/plant fresh weight and 15.47, 19.88 g/plant and 28.09,37.12 g/plant dry weight in the first and second seasons respectively.  

Table 2. Effect of Lawsonia inermis and Eucalyptus camaldulensis  leaves extracts on plant height

                           and number of  branches of lavandula dentata plants during 2012 and 2013 seasons

These results may due to the presence of the secondary metabolites, flavonoids, tannins, phenols and anthraquinones in Lawsonia inermis aqueous extract, the plant extracts may be considered as good sources of natural antioxidants for medicine uses (Arulpriya and Lalitha, 2012). These results are in harmony with (Muhammed, 2005; Tariqu et al., 2010).

4.Volatile oil percentage

Data in Table (4) showed that, all plants died when they were  treated with the pathogenic fungus alone  (control). Essential oil percentage of  Lavandula dentata fresh  herb were significantly responded to plant  extracts (natural fungicides)  in the first season at the  two cuts . The high concentration of Lawsonia inermis aqueous extract (100g/L) had a highest fungitoxic activity against Lavandula dentata root rot, which gave the highest oil percentage the values were 0.367 and 0.427% at the first and second cuts in the first season respectively. While in the second season the differences between the treatments were not significant. The lowest volatile oil percentage was obtained when the plants were sprayed with Eucalyptus camaldulensis aqueous extract at 50 g/L in the two seasons.The increment in volatile oil percentage may be due to medicinal and aromatic plants are the sources of natural fungicides because plants produce secondary metabolites such as phenols, phenolic acids, quinones, flavones, flavonoids, flavonols, tannins, coumarins and essential oils. These  groups have antimicrobial effect against pathogenic microorganisms (Das et al., 2010).

  5. GLC analysis of essential oil

The GLC analysis were carried out on the essential oil of Lavandula dentata plants of two treatments in the first cut of first season, Eucalyptus camaldulensis aqueous extract (50g/L) and Lawsonia inermis aqueous extract  (100 g / L). Data were recorded in Table (5) and Figures (1-2) revealed that, 1,8- Cineol was the main component. It was observed that content of 1,8- Cineol tended to increase up to 46.48 % in case of Lawsonia inermis aqueous extract  (100g/L).

Table 3. Effect of Lawsonia  inermis and Eucalyptus camaldulensis leaves extracts on herb fresh and dry weights  /plant (g) of   Lavandula   dentata plants during 2012 and 2013 seasons

           Table  4. Effect of Lawsonia inermis and Eucalyptus camaldulensis leaves extracts on volatile oil

                           percentage of Lavandula dentata plants during 2012 and 2013 seasons

Table 5. Effect of Lawsonia inermis and Eucalyptus camaldulensis leaves extracts on volatile oil components % of Lavandula dentata plants in the 1^St Season (1^St cut).

Figure 1. Chromatogram of Lavandula  dentata volatile oil distilled from  plants sprayed with Eucalyptus camaldulensis leaves aqueous extract at(50g/L)

Figure 2. Chromatogram of Lavandula  dentata volatile oil distilled from plants          sprayed with Lawsonia inermis leavesaqueous extract at (100g/L).