Control of Lasiodiplodia Theobromae (PAT) on Rhizophora Racemosa

Control of Lasiodiplodia theobromae (PAT) on Rhizophora racemosa using plants extracts *Ukoima, H.N ; Ikata,M and Pepple,G.A *Department of Forestry and Environment, Faculty of Agriculture , Rivers State University of Science and Technology,p.m.b.5080, Port Harcourt, Nigeria E-mail :[email protected] ABSTRACT Laboratory experiments were conducted to ascertain fungal isolates of Rhizophora racemosa .Studies on controlling L. theobromae using plant extracts was also carried out. Fungal pathogens were isolated by cutting the infected portions of the leaves and aseptically placing in glass Petri- dish for 7 days. Pathogenicity test was done in-situ with spore suspension inoculated on Rhizophora racemosa seedling leaves and observed for 15 days in the green house. Different concentrations ranging from (20%, 40%, 60%, 80% and 100%) of extract derived from the bark of Rhizophora racemosa, leaves of Aloe vera and Jatropha curcas were tested on the isolated fungi. The results showed that Lasidiplodia theobromae, Penicillium citrinum, Aspergillus niger and Paecilomyces lilacinus were isolated from the leaves of Rhizophora racemosa. Pathogenicity test revealed that Lasidiplodia theobromae was pathogenic on Rhizophora racemosa. Aloe vera and the bark of Rhizophora racemosa were inhibitory at 100% on Lasidiplodia theobromae (0.1645g and 0.2946g mycelial dry weight respectively). Jatropha curcas had les inhibitory effect at 80% concentration on the tested fungus (0.9118g mycelial dry weight). Biochemical test showed that Quinone, terpenoid and saponin from Aloe vera, bark of Rhizophora racemosa and Jatropha curcas respectively were suspected to account for the inhibition of the tested fungus (Lasidiplodia theobromae).

INTRODUCTION have been under utilized in the Niger Delta of Nigeria. They protect the shoreline and are particularly Red Mangrove thrives under a range of inter-tidal important in coastal areas that are subjected to major wetland conditions including high salinity levels from tropical storm tides during hurricanes and typhoons seawater to fresh water (Connell and Lowman 1989). (Basire PLAT, 2005). They tolerate a range of flooding, soil types and other physical site factors. Typically, they are common in Also, the mangrove serves as marine food chain. the middle to low inter-tidal zone above mean sea They play a vital role in protecting and supporting level, extending often along the seaward margin of marine food chains. The species create crucial mangrove stands. habitat needed for marine and estuarine invertebrate nesting grounds and food sources for wading birds They are medium to tall trees; they may reach 20- (Lucien-Brun, 1997). It is also a resource base for 50m in height. Although, commonly much shorter local inhabitants. It is a place they live and derive around 5-8m. Height growth is rapid after their means of livelihood (Ukoima et al, 2000). establishment on the substrate while food reserves are taken up from the hypocotyls of established Rhizophora spp have also been found as good seedlings. They tend to be of shorter statue and material for tannin and dyes. Extractives derived from more spreading in shape on the seaward edge of mangrove barks and hypocotyls for local industries stands or in sites of higher salinity. Taller, single include tannin and dyes. Tannin is a phenotic stemmed trees are found just behind the seaward substance used for the processing of leather, and to edge of stand (Connell and Lowman, 1989). produce dyes ranging from red, brown to black. Its importance is usually seen in the production of In Niger Delta, three red mangrove species are viscose rayon for all normal textile needs, tyre cords, recognized; Rhizophora mangle, Rhizophora industrial pulp (Bawayan, 1971). harrisonii and Rhizophora racemosa. Rhizophora harrisonii is a hybrid of the other two species (Wilcox- Some mangrove species have medical and Enwaraye, 1985). Mangroves are very important but pharmaceutical value. Red mangrove bark is used to Am. J. Biotechnol. Mol. Sci., 2013, 3(1): 1-7

treat angina, boils and fungal infections. The leaves various methods used to control plant diseases, use and bark have been used as an antiseptic and to of chemical fungicides is very common and very treat diarrhea, dysentery, fever, malaria and leprosy. effective. However in view of the complexities arising For example, it is used to cure throat cancer with from the use of chemical pesticides, such as harmful gargles of mangrove bark (Bandaranayale, 1999). effect on environment and non-target organisms including man, domestic animals, beneficial insects, It is a source for timber production. The wood of red wild/life, the use of biocides has provided a very mangrove is widely used for structural components of promising alternative and less hazardous method for traditional homes. For example, poles, beams, plant diseased control. flooring, well-cladding, rafters and others such as fencing, cabinet works, tool handles and boat anchor This research is therefore aim at isolating and etc. (Kohlmeyer, 1971). identifying fungi associated with leaf spot and mangrove seedlings and to confirm their Red mangroves serve as habitat for a wide range of pathogenicity. To evaluate the effects of some plant terrestrial and arboreal wild life. It provides shelter extracts such as Jatropha curcas, Aloe vera, bark of and food for a number of associated Fauna, including Rhizophora racemosa on Lasiodiplodia theobromae birds, small animals, shell fish, and other marine life. under in-vitro conditions. They stabilize soils with their network of sturdy overlapping prop roots which deepen water MATERIALS AND METHODS movement and promote sedimentation in areas that Location/Climate of Study Area: Laboratory might otherwise be eroded (Mehlig, 2006). experiments were carried out at the Rivers State In the Niger Delta mangrove ecosystem of Nigeria, University of Science and technology, Faculty of fungal pathogens of mangrove plants have been Agriculture located at latitude 4.43oS and 4.54oN and reported by researchers, (Ukoima, 1996). These longitude 6.56oW and 7o.03E Fodiboye, (1990). The fungi include Lasiodiplodia theobromae, field experiment was conducted at Ogonokom Paecilomyces lilacinus, Colletotrichum, Gloesporides, mangrove swamp in Abua/odual Local Government Fusarium moniliforme, Aspergillus flavus, Aspergillus Area of Rivers State, Latitude 04.150S, and 7.20oN niger, Alternaria solani, Penicillium citrinum, and longitude 5.5oW and 90o301E in the rainforest Botryodiplodia theobromae, Phoma sp. Rhizopus zone of Nigeria. The study area lies in the zone of stolonifer e.t.c. These fungi were isolated from humid tropical climate which has two seasons. The Rhizophora racemosa, Rhizophora mangle, wet season extends from March to October and dry Rhizophora harrisonii and Avicennia africana season extends from November to February. The (Ukoima 1996; 2000; 2006; 2007). A similar work has mean annual rainfall varies between 400mm - also been done in southern Florida (Newell, 1976). 1700mm. annual temperature range from 21oC and Botryodiplodia theobromae is known to cause seed 29oC (Fodiboye, 1990). rot, wilting and necrosis on mangrove plant in India Isolation and Identification of Fungi: Diseased (Mishra, 2002). Pestalotia palmarium have been leaves were collected from Ogonokom area of identified as causal agent of leaf spot at Batel-nut in Abua/Odual Local Government Area from parent Bangladesh (Islam, 2001). plants of Rhizophora racemosa and surface sterilized Efficacy of some plant extracts and plant products with methylated spirit and rinsed in distilled water. against powdery mildew of pea has been The infected portions of the leaves were cut into experimentally demonstrated. Ajoen, a constituent of pieces (1cm2), then placed on sterile filter paper in garlic at 100ppm was found to be inhibitory to some glass Petri-dish and incubated at room temperature fungi namely Aspergillus and saprophytic (27  2oC) for 7 days. The hyphal tips were Talaromyces flavus. (Singh et al 2000). Rhizome transferred into Potato dextrose agar (PDA) plate to powder of ginger gives a good control as sulfex and obtain pure cultures. The organisms were identified Bavistin. (Singh,2004). Different plant extracts like on the basis of morphological characteristics such as neem (Azadirachate indica), Tulshi (Ocimum colony form and color, type of mycelium, fruiting sanctum), Garlic (Alium sativium) Aloe vera, Bitter bodies and spores (Singh, 2000 and Toussoun and leaf (Veronica amygdalina), Scent leaf Cocimum Nelson, 1988). gratussima etc. have been recommended for the control of Pestalotia spp. and other fungi (Pandey et al, 1983; Ukoima and Okah 2006). Among the

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Pathogenicity Test: A total of forty red mangrove (R. The plant materials were washed several times with racemosa) plants were used for this experiment. distilled water and dried at room temperature. 100g of Plants to be inoculated were chosen randomly from plant part were separately crushed to a powder using 35-65cm height saplings of red mangrove, collected sterilized mortar and pestle. These crushed from Abua/Odual Local Government Area. materials were extracted sequentially into 300ml Inoculations were done in green house seedlings and distilled water. Phyto-chemical screening test was the leaflets were enclosed with spore suspension in done for different extracts of different solvents. The plastic bags. Leaves to be inoculated were extracts thus obtained were subjected to preliminary disinfected with cotton wool, dipped in methylated phyto-chemical screening following the methodology spirit and rinsed in sterile distilled water. Leaves were of Harborne (1998) and Kokate (2001). wounded by gently scraping the cuticles with Qualitative Analysis: inoculating needle. Fungi were re-isolated from infected leaves and identified (Yuan and Mohammed, Test for Saponins:- Froth test for saponins was 1999). used. 1g of the sample was weighed into a conical flask in which 10ml of sterile distilled water was Effect of Plant Extracts on Fungi: Plant extracts added and boiled for 5 min. The mixture was filtered were prepared using leaves of Aloe-vera and 2.5ml of the filtrate was added to 10ml of sterile (barbadense), Jatropha curcas, and bark of distilled water in a test tube. The test tube was Rhizophora racemosa. Plant leaves were washed in stopped and shaken vigorously for about 30 seconds. methylated spirit for 3 minutes and then rinsed with It was then allowed to stand for half an hour. distilled water and sundried. Five hundred grammes Honeycomb forth indicated the presence of saponins. (500g) of the test plant part were blended into (500ml) distilled water and filtered using a muslin Test for Terpenoids:- 10mg of the extract was white cloth. The resulting extracts were taking as dissolved in 1ml of chloroform. This was followed by 100% concentration of plant extract. The following the filtration process. 1ml of acetic anhydride was concentration levels 20%, 40% 60%, 80%, 100% was added following the addition of 2ml of conc H2SO4. used. The experiment was in five replicates for each Formation of reddish violet colour indicated the treatment. presence of terpenoids. Potato dextrose broth (PDB) was poured into each Test for Quinones:- 5ml of the extract solution conical flask to make up the various concentrations. was hydrolysed with diluted conc. H2SO4 extracted 20ml of the concentrated plant extract prepared was with benzene. 1ml of dilute ammonia was added to pipetted into the conical flasks containing Potato it. Rose pink coloration indicated the presence of dextrose broth (PDB). A disk of Lasiodiplodia quinones. theobromae was inoculated into the various conical Quantitative Analysis: Fat free sample was flasks with a cork borer of 0.5cm diameter and prepared using; 2g of the sample were deffated with incubated at room temperature for 7 days. All the 100ml of diethyl either using a soxhlet apparatus for incubated conical flasks were constantly agitated to 2hrs. allow even distribution of the fungi. On the 7th day the mycelia were harvested by filtering through a Saponin Determination:- Each samples were o known mass of filter paper and then dried at 60 C for ground and 20g of each were put into a conical flask 24 hours. Potato dextrose broth (PDB) with pure and 100cm3 of 20% aqueous ethanol were added. fungi served as the control. (Srinivas, et al 1997) The samples were heated over a hot water bath for o Analyzing the Active Ingredient in the Plant 4hrs with continuous stirring at about 55 C. The Extract: Screening of some Phytochemical mixture was filtered and the residue re-extracted with constituents of the plant extract, (Aloe-vera, Jatropha another 100ml of 20% ethanol. The combined curcas and back of Rhizophora racemosa to identity extracts were reduced to 40ml over water bath at about 90oC. Then, the concentrate was transferred the active ingredient was done using standard into a 250ml separating funnel. 10ml Diethyl either procedures as described by Harborne (1998) and was added to the funnel and the mixture shaken Kokate (2001) at the Biochemistry laboratory of the vigorously. The aqueous layer was recovered while Rivers State University of Science and Technology (RSUST). the ether was discarded. The purification process was repeated.

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In addition, 30ml of n-butanol was added. The Pathogenicity Test: The result on Pathogenicity combined n-butanol extract was washed twice with indicated that Lasiodiplodia theobromae caused 5ml of 5% aqueous sodium chloride. The remaining necrotic lesion on the leaves of Rhizophora. solution was then heated in a water-bath. After racemosa (Plate 2). evaporation the samples were dried in the oven to a constant weight. The saponin content was calculated as percentage. (Obadoni and Ochuko, 2001). Determination of Quinone:- 2g of plant samples were defatted with 200ml of diethyl ether using soxhlet apparatus for 5hrs. The fat free samples were boiled with 300ml of chloroform for the extraction of the Quinone component for 30mins. Upon cooling the reaction mixture were basify with a saturated aqueous solution of sodium hydrogen carbonate. The reaction mixture were extracted into 50ml dichlotromethane and the organic layer were Plate 2: Seedling Showing Dark Brown spots caused dried over Na2 S04 and evaporated under reduced pressure. The extraction efficiency was calculated in by Lasiodiplodia theobromae percentage. (Pakulski and Budzianowski, 1996). Determination of Terpenoid:- 50g of the plant Effect of Plant Extracts on Fungal Pathogens: The extract were extracted with solvent combination of result on tables 1, 2 and 3 showed that 100% Aloe- methanol and water at room temperature for 24hr. vera and R. racemosa had the highest effect on the The solution were filtered using Whatman filter paper growth of Lasiodiplodia theobromae. Mycelial dry and the filtrate were then evaporated to 1/10 volume weight for the fungus was 0.1645g in Aloe vera and at 40oC. The evaporated filtrate was acidified with 2m 0.2946g in R. racemosa. The highest effect for sulphuric acid (pH 0.89) followed by chloroform Jatropha curcas on the fungus was in 80% with extraction (three times the volume), stirred and mycelial dry weight of 0.8610g. The least effect was allowed to stand in a separatory funnel. Out of the in the control for all treatments. These results are two layers formed, the non-aqueous layer was taken also presented in figures 1,2 and 3. These results and evaporated till dryness. were not significantly different at P > 0.05 using DMRT. The dried extract contained terpenoids which were further weighed and calculated as percentage (Majaw Table 1: Effect of Aloevera Extracts on Lasiodiplodia theobromae growth (g) et al, 2009). % Aloe vera concentration Organism RESULTS

Isolation of Pathogen: The fungal pathogens a isolated and identified, from the leaves of Rhizophora 20% 0.5332 racemosa were; Lasiodiplodia theobromae (Plate 1), 40% 0.6352a Paecilomyces lilacinus, Aspergillus niger and Penicillium citrinum 60% 0.2990b

80% 0.5654a

100% 0.1645b

Control 3.7532c

Values in each column with the same super script are not significantly different at P>0.05 using DMRT.

Plate 1:Pure culture of Lasiodiplodia theobromae

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Table 2: Effect of Jatropha curcas Extracts on Quantitative Phytochemical Analysis: The result Lasiodiplodia theobromae growth (g) on table 4 showed that the bark of Rhizophora % Jatropha curcas Organism rasemosa contained 0.08 ± 0.10; 0.00 for Quinone concentration and terpenoid 18.12 ± 0.18. Jatropha curcas 20% 1.4346a contained 0.02 ± 0.01 saponins; 0.00 for both quinone and terpenoid. Aloe-vera had 0.04 ± 0.01 a 40% 1.3381 saponins, 20.10 ± 0.12 Quinone and 0.00 terpenoid. a 60% 1.0305 Table 5: Weight (%) of Bioactive Secondary 80% 0.8610b Compounds in bark of Rhizophora racemosa, Aloe- vera and Jatropha curcas (Mean  SE) 100% 0.9118b Plants Saponins Quinone Terpenoid Control 3.9491c Bark of 0.08 ± 0.10 0.00 18.12 ± 0.18 Values in each column with the same super script are Rhizophora not significantly different at P>0.05 using DMRT. racemosa (mg/g) Table 3: Effect of Rhizophora racemosa Jatropha curcas 0.02 ± 0.01 0.00 0.00 Extracts on Lasiodiplodia theobromae (mg/g) growth (g) Aloe-vera (mg/g) 0.04 ± 0.01 20.10 ± 0.00 % Rhizophora racemosa Organism 0.12 concentration

20% 0.9009a DISCUSSION 40% 0.8425a The fungal Pathogens isolated and identified from 60% 0.4126b leaf of Rhizophora racemosa were Lasiodiplodia 80% 0.5208b theobromae, Paecilomyces lilacinus, Aspergillus 100% 0.2946c niger, and Penicillium citrinum based on their

c morphology and conidial characteristic. Control 4.4120 Botriyodiplodia theobromae was found to be Values in each column with the same super script are pathogenic on excised leaves sapling of Rhizophora not significantly different at P>0.05 using DMRT. racemosa in the green house. Ukoima (1996; 2000; Qualitative Phytochemical Analysis: 2006 ) working on mangrove leaves isolated these fungal pathogen from mangrove plant. Misral et al, Phytochemical analysis of the leaf extracts of Aloe- (2002) also observed that Lasiodiplodia theobromae vera, Jatropha curcas, and bark of Rhizophora racemosa is shown in table 4. The leaf extract of Aloe caused seed rot, wilting and necrosis on mangrove plants in India. vera showed the presence of Quinones and Saponins while the bark of Rhizophora racemosa The results on plant extracts showed that Aloe-vera extracts showed the presence of Terpenoids and and R. racemosa at 100% had the highest effect on Saponins. The leaf extract of Jatropha curcas the growth of Lasiodiplodia theobromae. Mycelia dry showed the absence of Terpenoids, Quinones and weight for the fungus was 0.1645g in Aloe vera and saponins. 0.2946g in Rhizophora racemosa. The least effect Table 4: Phytochemical Screening of Aloe Vera, was in the control for all treatments. (Srinivas et al, Jatropha curcas and bark of Rhizophora racemosa 1997) reported that Aloe vera extract performed best against Pestalotiopsis longiseta in-vitro on tea plants Phytochemical Aloe Jatropha Bark of (Camellia sinensis) and blight of sunflower (Alternaria vera curcas Rhizophora racemosa alternate) Chuku, (2006) also reported the anti fungal properties of Aloe vera gel on Aspergillus niger. Saponins + - + Other works that supported this finding were those of Terpenoids - - + Kishore et al (1982), and Naidu and John (1981) who Quinones + - - reported the effective fungicidal potential extracts of Aloe vera on Ranunculus sclereratus. Oil extract + = Presence from Caesulia axillaries was reported to be superior - = absence to eight synthetic fungicides and it exhibited the

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strongest toxicity against the leaf spot disease of rice measure for Lasiodiplodia theobromae. Hulchings et caused by Helminthosporium oryzde (Pandey et al, al, (1996) reported that saponins, terpernoid and 1983). quinone compounds were useful in pharmaceutical companies as anti-microbial, anti-fungal, anti- Chandrasekaran et al (2009) reported that matured inflammatory, anti-oxidative damage diseases etc leaves and bark of L.racemosa and R. apiculata exhibited different degree of growth inhibition against CONCLUSION Lasiodiplodia theobromae. Also, Garlic extract at 4 Lasiodiplodia theobromae,is pathogenic on and 5% concentrations were reported as the best Rhizophora racemosa. Aloe-vera and bark of plant extracts, which inhibited 100% of radial growth Rhizophora racemosa extract were found to be of Pestalotia palmarium on Betal nut (Areca catachi) effective botanical to control Lasiodiplodia in-vitro. theobromae. Secondary compounds such as Result of qualitative screening of plants extract, bark Quinones, saponins and Terponoids were found to of Rhizophora racemosa showed the presence of be the phytochemical constituents responsible for the terperniod and saponins, quinone in Aloe-vera while effect. Jatropha curcas showed none of these phyto- RECOMMENDATION chemicals. Barnabas (1988) reported the presence of terpernoid, alkaloid, flavounoid, tannin, stenoids in Aloe-vera, bark of Rhizophora racemosa extracts, L. racemosa and Avicennia marine. Also, the and the use of natural antagonistic organism may be presence of polyphenols and small quantity of recommended for field treatments of fungal diseases saponins are reported as the major constituents of of Rhizophora spp. These plant materials are readily Jatropha curcas (Dessisa, 1998). The result on accessible and available in the localities and cheap to quantitative phyto-chemical analysis showed that the procure. They are environmentally friendly, useful to bark of Rhizophora racemosa contained 0.08 ± the resource – poor, educationally poor rural dwellers 0.10%; 0.00 for Quinone and terpenoid 18.12 ± who use mangrove in their daily needs. 0.18%. Jatropha curcas contained 0.02 ± 0.01% Finally, it is necessary to synthesize these active saponins; 0.00 for both quinone and terpenoid. Aloe- vera had 0.04 ± 0.01% saponins, 20.10 ± 0.12 ingredients from the plants for minimizing Lasiodiplodia theobromae on mangroves. Quinone and 0.00 terpenoid. Quantitatively, phytoalexins compound have been demonstrated on REFERENCES a wide variety of plants. Terpernoids example Bandaranayale, W.M. Economics, Traditional and limonene, a-pinene compound from Zingerber Medicinal uses of mangrove, Australian Institute of cassumunar effectively inhibited the growth of Botiytic Marine Science, Aim Report Townsville Australia. 1999 fabae of pea (Majaw and Moirangthen, 2009). Also, Barnabas, C.G. Antibacterial Activity of Flavonoids of some carvone from Clerodendro colebrookkianum walp Medicinal Plants. Fitoterapia 3:508-10. 1988 leaves inhibited the growth of Rhodococcus Basire, J.D., Dahdou-Guebas, F.J., Koedam, L.P. Nitto, enythropolis (Morrish et al, 2008). Quinone example D.D. How Effective were Mangrove as a Defense Dichlone compound has been used effectively against the Recent Tsunami, Current Biology Vol. against apple scab, peach leaf curl Thomson (1997), 15:443-447. 2005 Torssel (1983) reported that quinone compound Bawayan, B.O. Cellulose Derivatives II. Cellulose Xanthate. polyketide, shikimic acid are known bio-chemical Forpridecom Tech, Note 1004:2. 1971 defense for plant, polyketide suppresses Plumbago Chandrasekaran, M. Kannathasan, K. Venkatesalu V. auriculata. Saponins compound tomatin were Prabhakar K. Antibacterial activity of some salt marsh effective inhibitory on Fusarium oxysporium. halophytes and mangrove plants against Methicillin Hulchings et al, (1996). resistant staphylococcus aureus. World J. Microbiol Biotechnol 2: 155-60. 2009 The result of the study showed the presence of Chuku, E.C. Controlling Aspergillus niger in-vitro using saponins, terpernoid and quinone. Therefore, these crude extracts of Aloe vera in Rivers State. Journal of plants can be a potential source of useful medicinal Research in Biosciences. Vol 2 (3) 44-48. 2006 values. Also, table 5 indicated that there were more Connell, J. H and M. D. Lowman . Low-diversity Tropical concentration of total quinone (20.100.12%) in Aloe- Rain Forests: some Possible Mechanisms for their vera and Rhizophora racemosa (18.120.18%). Existence. Am Nat 134-145. 1989 Since these bioactive compounds are known to have Dessisa, D.A. A Preliminary Economic Evaluation antifungal properties. It may be an effective control Medicinal Plants in Ethiopia: Trade, Volume and price,

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