Antibacterial activities of sawdust and stem bark of Sasswood tree (Erythrophleum suaveolens, Guill. & Perr. Brenan, 1917) extracts against selected wood bacteria

This study assessed the antibacterial properties of sawdust and stem bark of Erythrophleum suaveolens extracts on selected wood bacteria. Erythrophleum suaveolens samples were collected, dried and macerated by dissolving 1 Kg and 0.60 Kg of stem bark and sawdust respectively into 1 L of n-hexane, ethyl acetate and methanol. Sporfloxacin ciprofloxacin and cefuroxine antibiotics were used as control. The mixture was left for 24 hours then filtered and the filtrates evaporated to dryness. Qualitative phytochemical screening, zone of inhibition, minimum inhibitory and Bactericidal Concentrations (MIC/MBC) were determined according to standard methods. Tannins, steroids, saponins, glycosides, flavonoids, carbohydrates anthraquinones and alkaloids phytochemicals were present in E. suaveolens extracts. Zone of inhibition (32 – 37 mm) of antibiotics on test bacteria compared favourably with 17 – 24 mm of E. suaveolens extracts. Erythrophleum suaveolens ethyl acetate and methanol E. suaveolens extracts inhibited Staphylolococus aureus, Ralstonia solanacearum, Proteus mirabilis, Enterococcus faecium and Acidobacterium capsulatum growth at MIC of 10 mg/mL and n-hexane extracts at 20 mg/mL. At MBC of 20 mg/mL methanol stem bark extract completely killed most test bacteria. Methanol extracts were the most active extracts. The study has shown that E. suaveolens extracts can be explored in the control of plant diseases caused by test bacteria in the study. 
Key words: Antibacterial, E. suaveolens, extract, phytochemicals, zone of inhibition


Introduction
Wood is an essential and renewable natural resource. It is common in our daily lives with high economic importance and used in many areas such as in furniture and wood-frame houses. It is natural resources for the production of books, newspapers, textile fabrics; and magazines. It has application in bridge construction and railroad ties. Wood is also used as utility poles, fence posts and source of organic chemicals. Wood and wood products store carbon, therefore helping to reduce carbon dioxide in the atmosphere (Durbak et al. 1998).
Despite its remarkable usefulness, wood is decomposed by a range of biological agents, including insects, bacteria and fungi (Ekhuemelo et al., 2019). Bacteria are known as the principal degraders of wood and wooden structures mainly those recovered from oxygen-deficient environments (Singh et al., 2016). The relationship between bacteria and wood has been known since as six decades ago (Clausen, 1996). However, not much interest has been given to the activities of bacteria on deterioration of wood in relation to the extent of deterioration caused by wood degrading fungi. Wood is inhabited by bacteria which are associated with wood degradation and could have an indirect influence on wood decay process. Bacteria can affect wood permeability, damage wood structure, or work in association with some bacteria and soft-rot fungi to dispose wood to fungal attack (Clausen, 1996).
Bacteria infecting wood may affect the permeability resulting in loss of strength, damage and destruction of wood structure (Greaves, 1971). Bacterial population varies depending on the state of wood decay as bacterial communities increase as the wood decomposition progresses (Hoppe et al., 2015). Bacteria that attack wood structure known as tunneling bacteria, degrade wood cell wall from the area of cell lumen where they start to multiply (Clausen, 1996).These bacteria are usually present in terrestrial and aquatic environments and can inhabit and degrade wood found in oxygenated environments and under conditions with very low levels of oxygen (Singh et al. 2016).
Erythrophleum suaveolens (Guill. & Perr. Brenan) tree belongs to the family Fabaceae. It is a very common medicinal plant in Africa. The tree bark is used as arrow poisons while the leaves are used as fish poison. Various preparations of the bark have been used to treat inflammations caused by Filaria, heart failure, headache, and body pains. The wood is appropriate for furniture, flooring, dock work, railway sleepers, turnery, construction, harbour and bridges, boat building and wheel hubs (Burkill, 1985;Akinpelu et al. 2012).
Synthetic chemicals such as creosote, Copper Chrome Arsenic (CCA), Alkaline Copper Quaternary (ACQ) and synthetic pyrethroids are persistent and toxic to the environment and human. The uses of bio-pesticides from this study are eco-friendly, biodegradable, cost effective, less toxic and readily available for wood preservation in Nigeria.
This has resulted in the need for the use of bioactive compounds as alternatives (Clausen, 1996). Although extracts from this plant have been used for medicinal purposes, there is dearth of information on its antibacterial activities on wood tunneling and erosion bacteria. This study therefore focused on assessing the effect of the sawdust and stem bark extracts in the management of wood degrading bacteria.  (Uleh et al. 2017). The samples collected were air dried and the stem bark crushed prior to solvent extraction.

Plant collection and preparation for extraction
Crude extraction of stem bark and sawdust The extraction of E. suaveolens stem bark and sawdust was done by macerating 1 Kg and 0.60 Kg of the stem bark and sawdust into 1 L of n-hexane separately for 24 hours and filtered. The residue from the hexane extraction was equally macerated again with ethyl acetate and methanol respectively within 24 hours. The extracts were filtered and dried before antibacterial test.
Phytochemical screening of stem bark and sawdust of Erythrophleum suaveolens Phytochemical tests for alkaloids, tannins, steroids, saponin, cardiac glycosides, anthraquinones, flavonoids, carbohydrates and glycosides of stem bark and sawdust samples were performed using standard methods adopted by Ekhuemelo et al. (2019).

Antibacterial Screening and Sensitivity on test bacteria
Antibacterial screening was carried out on Staphylolococus aureus, Ralstonia solanacearum, Pseudomonas syringae, Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus faecium, Bacillus subtilis, Agrobacterium tumefaciens, Actinobacterium sp. and Acidobacterium capsulatum using diffusion method described by Akinpelu and Onakoya (2006). The initial concentration of extracts was obtained by dissolving 0.4 g extract in 10 mL Dimethyl sulfoxide to give a concentration of 40 mg/mL. Media were prepared in accordance with producer's guide and purified at 121 o C for a period of 15 minutes. The prepared media was poured into germ-free Petri dishes and left to cool and harden. Muller Houston agar was seeded with a standard inoculum (0.1 mL) of the test bacteria spread uniformly on the surface of the medium with the aid of a disinfected swab. Cork borer measuring 6 mm in diameter was utilized to cut a well at the middle of each injected medium. Thereafter, 0.1 mL sample solution of 40 mg/mL concentration was introduced into the well of the inoculated medium and incubated at 37 o C for 24 hours. The media plates were observed for zone of inhibition (ZOI) of bacteria growth. The ZOI were measured and readings were recorded in millimeters.

Determination of minimum inhibitory and Bactericidal Concentrations (MIC/MBC)
The MIC of the Erythrophleum suaveolens extracts was determined using broth dilution technique (Andrew, 2001). Standardized suspensions of the test bacteria were inoculated into plates containing antibiotics (sporfloxacin, ciprofloxacin and cefuroxine) as control. Five concentrations of 40 mg/mL, 20 mg/mL, 10 mg/mL, 5 mg/mL and 2.5 mg/mL of the crude extracts were used to determine the minimum bactericidal concentration (MBC). The inoculated plates were incubated for 24 hours. Plates that showed no bacterial growth were recorded for MIC.

Data Analysis
Data on zone of inhibition was analyzed by one-way Analysis of Variance for significance differences. Mean separation was done using Duncan New Multiple Range Test (DNMRT) where significant means were recorded.

Results
Phytochemical screening of Erythrophleum suaveolen stem bark showed the presence of tannins, steroids, saponins, glycosides, flavonoids, carbohydrates anthraquinones and alkaloids. Cardiac glycosides were not present in the stem bark extract of E. suaveolen. The result also revealed that the sawdust extract contained steroids, flavonoids, cardiac glycosides and carbohydrates while tannins, saponins, glycosides, anthraquinones and alkaloids were absent. to ciprofloxacin and cefuroxine they were sensitive to Sporfloxacin at ZOI of 32 mm and 30 mm, respectively. There was no significant difference (p>0.05) in the Zone of inhibition among the antibiotics.
The phytochemicals found in Erythrophleum suaveolens plant implies that they could be important bioactive agents against wood bacteria.
The Zone of inhibition (32 -37 mm) of antibiotics on test bacteria in this study compared favourably with 17 -24 mm of Erythrophleum suaveolens extracts. Guevara, (2005) reported that when zone of inhibition of pesticides are < 10 mm; 10 -13 mm, 14 -19 mm, and >19 mm they are said to be inactive, partially active, active and very active, respectively. It therefore implies that the crude extracts of stem bark and sawdust of E. suaveolens in this study ranged from been active to very active, while the antibiotics were very active. Aiyegoro et al. (2007) obtained lower zone of inhibition of 10,9,10,12,9,14,11,13 (Abubakar, 2010).
This study has demonstrated that Erythrophleum suaveolens extracts can be explored in the control of wood deterioration caused by the bacteria screened in this study such as wood-decay, cellulolytic and ligninmodifying activities, bacterial wilt, leaf-spot, soft rot and bark canker diseases.

Conclusion
This study has shown some bioactive phytochemicals such as tannins, steroids, saponins, glycosides, flavonoids, carbohydrates anthraquinones and alkaloids as been present in Erythrophleum suaveolen. Erythrophleum suaveolens methanol extracts were more active in comparison with ethyl acetate and n-hexane extracts. The finding from the study has revealed that Erythrophleum suaveolens extracts could control some bacteria of plants and animal disease.